Phytate polynucleotides and methods of use

ABSTRACT

This invention relates to newly identified polynucleotides and polypeptides in the phytic acid biosynthetic pathway, variants and derivatives of same; methods for making the polynucleotides, polypeptides, variants, derivatives and antagonists. In particular the invention relates to polynucleotides and polypeptides of the inositol 1,3,4-trisphosphate 5/6-kinase gene family. In particular this invention relates to using the newly identified polynucleotides and polypeptides to modulate the phytic acid biosynthesis in such a way as to decrease phytate and/or increase non-phytate phosphorous, especially in corn or soy animal feedstuffs.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Application SerialNo. 60/325,308 filed Sep. 27, 2001, which is herein incorporated byreference.

TECHNICAL FIELD

[0002] The present invention relates to the field of animal nutrition.Specifically, the present invention relates to the identification anduse of genes encoding enzymes involved in the metabolism of phytate inplants and the use of these genes and mutants thereof to reduce thelevels of phytate, and/or increase the levels of non-phytate phosphorusin food or feed.

BACKGROUND OF THE INVENTION

[0003] The role of phosphorous in animal nutrition is well recognized,it is a critical component of the skeleton, nucleic acids, cellmembranes and some vitamins. Though phosphorous is essential for thehealth of animals, not all phosphorous in feed is bioavailable.

[0004] Phytates are the major form of phosphorous in seeds, for examplephytate represents about 60-80% of total phosphorous in corn andsoybean. When seed-based diets are fed to non-ruminants, the consumedphytic acid forms salts with several important mineral nutrients, suchas potassium, calcium, and iron, and also binds proteins in theintestinal tract. These phytate complexes cannot be metabolized bymonogastric animals and are excreted, effectively acting asanti-nutritional factors by reducing the bioavailability of dietaryphosphorous and minerals. Phytate-bound phosphorous in animal excretaalso has a negative environmental impact, contributing to surface andground water pollution.

[0005] There have been two major approaches to reducing the negativenutritional and environmental impacts of phytate in seed. The firstinvolves post-harvest interventions, which increase the cost andprocessing time of feed. Post-harvest processing technologies removephytic acid by fermentation or by the addition of compounds, such asphytases.

[0006] The second is a genetic approach. One genetic approach involvesdeveloping crop germplasm with heritable reductions in seed phytic acid.While some variability for phytic acid was observed, there was no changein non-phytate phosphorous. Further, only 2% of the observed variationin phytic acid was heritable, whereas 98% of the variation wasattributed to environmental factors.

[0007] Another genetic approach involves selecting low phytate linesfrom a mutagenized population to produce germplasm. Most mutant linesare a loss of function, presumably blocked in the phytic acidbiosynthetic pathway, therefore low phytic acid accumulation will likelybe a recessive trait. In certain cases, this approach has revealed thathomozygosity for substantially reduced phytate proved lethal.

[0008] Another genetic approach is transgenic technology, which has beenused to increase phytase levels in plants. These transgenic planttissues or seed have been used as dietary supplements.

[0009] The biosynthetic route leading to phytate is complex and notcompletely understood. Without wishing to be bound by any particulartheory of the formation of phytate, it is believed that the synthesismay be mediated by a series of one or more ADP-phosphotransferases,ATP-dependent kinases and isomerases. A number of intermediates havebeen isolated including, for example, monophosphates such asD-myo-inositol 3-monophosphate, diphosphates (IP₂s) such asD-myo-inositol 3,4-bisphosphate, trisphosphates (IP₃S) such asD-myo-inositol 3,4,6 trisphosphate, tetraphosphates (IP₄S) such asD-myo-inositol 3,4,5,6-tetrakisphosphates, and pentaphosphates (IP₅s)such as D-myo-inositol 1,3,4,5,6 pentakisphosphate. The phosphorylationof the IP5 to IP₆ is found to be reversible. Several futile cycles ofdephosphorylation and rephosphorylation of the IP₅ and IP₆ forms havebeen reported as well as a cycle involvingglucose-6-phosphate->D-myo-inositol 3-monophosphate->myo-inositol; thelast step being completely reversible, indicating that control ofmetabolic flux through this pathway may be important.

[0010] Based on the foregoing, there exists the need to improve thenutritional content of plants, particularly corn and soybean byincreasing non-phytate phosphorous and reducing seed phytate. Thisinvention provides tools and reagents that allow the skilled artisan, bythe application of, inter alia, transgenic methodologies to influencethe metabolic flux in respect to the phytic acid pathway.

SUMMARY OF THE INVENTION

[0011] Inositol 1,3,4-trisphosphate 5/6-kinases (ITPK) are involved inthe phytate biosynthetic pathway. This invention provides nucleic acidsand proteins related to inositol 1,3,4-trisphosphate 5/6-kinases as wellas recombinant expression cassettes and methods to modulate the level ofinositol 1,3,4-trisphosphate 5/6-kinases in host cells, transgenicplants and seeds. The invention also provides the host cells, transgenicplants and transgenic seeds produced by these methods. The inventionforesees using these nucleic acids or polypeptides, or variants thereof,to modulate the flux through the phytic acid biosynthetic pathway inorder to improve the nutritional quality of feed, corn and soy inparticular, and to reduce the environmental impact of animal waste bycreating seed with higher available phosphorous or lower phytate levels.

DETAILED DESCRIPTION OF THE INVENTION

[0012] Definitions

[0013] Units, prefixes, and symbols may be denoted in their SI acceptedform. Unless otherwise indicated, nucleic acids are written left toright in 5′ to 3′ orientation; amino acid sequences are written left toright in amino to carboxy orientation, respectively. Numeric rangesrecited within the specification are inclusive of the numbers definingthe range and include each integer within the defined range. Amino acidsmay be referred to herein by either their commonly known three lettersymbols or by the one-letter symbols recommended by the IUPAC-IUBBiochemical Nomenclature Commission. Nucleotides, likewise, may bereferred to by their commonly accepted single-letter codes. Unlessotherwise provided for, software, electrical, and electronics terms asused herein are as defined in The New IEEE Standard Dictionary ofElectrical and Electronics Terms (5th edition, 1993). The terms definedbelow are more fully defined by reference to the specification as awhole.

[0014] The term “isolated” refers to material, such as a nucleic acid ora protein, which is: (1) substantially or essentially free fromcomponents which normally accompany or interact with the material asfound in its naturally occurring environment or (2) if the material isin its natural environment, the material has been altered by deliberatehuman intervention to a composition and/or placed at a locus in the cellother than the locus native to the material.

[0015] As used herein, the term “nucleic acid” means a polynucleotideand includes single or multi-stranded polymers of deoxyribonucleotide orribonucleotide bases. Nucleic acids may also include fragments andmodified nucleotides. Therefore, as used herein, the terms “nucleicacid” and “polynucleotide” are used interchangably.

[0016] As used herein, “inositol 1,3,4-trisphosphate 5/6-kinasepolynucleotide” or “ITPK polynucleotide” means a polynucleotide encodinga polypeptide with inositol 1,3,4-trisphosphate 5/6-kinase activity, ora polynucleotide capable of modulating the expression of mRNA or proteinin a host cell. The term is also inclusive of fragments, variants,homologues, alleles or precursors with the any one of the above statedfunctions.

[0017] As used herein, “ITPK” means inositol 1,3,4-trisphosphate5/6-kinase in regards to any nucleic acid or polypeptide, or theassociated functional activity.

[0018] As used herein, “polypeptide” means proteins, protein fragments,modified proteins (e.g., glycosylated, phosphorylated, or othermodifications), amino acid sequences and synthetic amino acid sequences.The polypeptide can be modified or not. Therefore, as used herein,“polypeptide” and “protein” are used interchangably.

[0019] As used herein, “inositol 1,3,4-trisphosphate 5/6-kinasepolypeptide” or “ITPK polypeptide” which is capable of phosphorylatingan appropriate inositol phosphate substrate and refers to one or moreamino acid sequences, in modified or unmodified form. The term is alsoinclusive of active fragments, variants, homologs, alleles or precursors(e.g., preproproteins or proproteins) or activity thereof.

[0020] As used herein, “plant” includes plants and plant parts includingbut not limited to plant cells and plant tissues such as leaves, stems,roots, flowers, pollen, and seeds.

[0021] As used herein, “promoter” includes reference to a region of DNAupstream from the start of transcription and involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.

[0022] By “fragment” is intended a portion of the nucleotide sequence ora portion of the amino acid sequence and hence protein encoded thereby.Fragments of a nucleotide sequence may encode protein fragments thatretain the biological activity of the native nucleic acid, functionalfragments. Alternatively, fragments of a nucleotide sequence that can beuseful as hybridization probes may not encode fragment proteinsretaining biological activity. Thus, fragments of a nucleotide sequenceare generally greater than 25, 50, 100, 150, 200, 250, 300, 350, 400,450, 500, 600, or 700 nucleotides and up to and including the entirenucleotide sequence encoding the proteins of the invention. Generallythe probes are less than 1000 nucleotides and often less than 500nucleotides. Fragments of the invention include antisense sequences usedto decrease expression of the inventive polynucleotides. Such antisensefragments may vary in length ranging from greater than 25, 50, 100, 200,300, 400, 500, 600, or 700 nucleotides and up to and including theentire coding sequence.

[0023] By “functional equivalent” as applied to a polynucleotide or aprotein is intended a polynucleotide or a protein of sufficient lengthto modulate the level of ITPK protein activity in a plant cell. Apolynucleotide functional equivalent can be in sense or antisenseorientation.

[0024] By “variants” is intended substantially similar sequences.Generally, nucleic acid sequence variants of the invention will have atleast 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequenceidentity to the native nucleotide sequence, wherein the % sequenceidentity is based on the entire sequence and is determined by GAP 10analysis using default parameters. Generally, polypeptide sequencevariants of the invention will have at least about 60%, 65%, 70%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity tothe native protein, wherein the % sequence identity is based on theentire sequence and is determined by GAP 10 analysis using defaultparameters. GAP uses the algorithm of Needleman and Wunsch (J. Mol.Biol. 48:443-453, 1970) to find the alignment of two complete sequencesthat maximizes the number of matches and minimizes the number of gaps.

[0025] As used herein “transformation” may include stable transformationand transient transformation. Unless otherwise stated, “transformation”refers to stable transformation.

[0026] As used herein “stable transformation” refers to the transfer ofa nucleic acid fragment into a genome of a host organism (this includesboth nuclear and organelle genomes) resulting in genetically stableinheritance. In addition to traditional methods, stable transformationincludes the alteration of gene expression by any means includingchimeraplasty or transposon insertion.

[0027] As used herein “transient transformation” refers to the transferof a nucleic acid fragment or protein into the nucleus (orDNA-containing organelle) of a host organism resulting in geneexpression without integration and stable inheritance.

[0028] “ITPK enzyme-binding molecule”, as used herein, refers tomolecules or ions which bind or interact specifically with phytatebiosynthetic enzyme polypeptides or polynucleotides of the presentinvention, including, for example enzyme substrates, cofactors,antagonists, inhibitors, cell membrane components and classicalreceptors. Binding between polypeptides of the invention and suchmolecules, including binding or interaction molecules may be exclusiveto polypeptides of the invention, or it may be highly specific forpolypeptides of the invention, or it may be highly specific to a groupof proteins that includes polypeptides of the invention, or it may bespecific to several groups of proteins at least one of which includes apolypeptide of the invention. Binding molecules also include antibodiesand antibody-derived reagents that bind specifically to polypeptides ofthe invention.

[0029] “High phosphorous transgenic”, as used herein, means an entitywhich, as a result of recombinant genetic manipulation, produces seedwith a heritable decrease in phytic acid percentage and/or increase innon-phytate phosphorous percentage as compared to a corresponding plantthat has not been transformed.

[0030] “Phytic acid”, as used herein, means myo-inositol tetraphosphoricacid, myo-inositol pentaphosphoric acid or myo-inositol hexaphosphoricacid. As a salt with cations, phytic acid is “phytate”.

[0031] “Non-phytate phosphorous”, as used herein, means total phosphorusminus phytate phosphorous.

[0032] “Non-ruminant animal” means an animal with a simple stomachdivided into the esophageal, cardia, fundus and pylorus regions. Anon-ruminant animal additionally implies a species of animal without afunctional rumen. A rumen is a section of the digestive system wherefeedstuff/food is soaked and subjected to digestion by microorganismsbefore passing on through the digestive tract. This phenomenon does notoccur in a non-ruminant animal. The term non-ruminant animal includesbut is not limited to humans, swine, poultry, cats and dogs.

Nucleic Acids

[0033] Inositol 1,3,4-trisphosphate 5/6 kinases (ITPKs) are involved inthe phytate biosynthetic pathway. The enzymes of the present inventionhave a broader substrate specificity than expected and can phosphorylateseveral IP3 and IP₄ inositol phosphate species using adenosinetriphosphate (ATP) as the phosphate donor, resulting in the productsadenosine diphosphate (ADP) and a phosphorylated inositol phosphate. Itis expected that this enzyme acts sufficiently downstream ofmyo-inositol in the phytate pathway so that modulation of this enzymemay decrease phytate accumulation without significantly impactingmyo-inositol levels. The sequences of the present invention havehomology throughout the entire sequence to known ITPK nucleic acids andproteins. It is expected that modulating the expression and/or level ofthe nucleic acids of the present invention will modulate the phytatebiosynthetic pathway providing methods to increase availablephosphorous, decrease phytate and/or decrease polluting phytate-boundphosphorous in animal waste.

[0034] The isolated nucleic acids of the present invention can be madeusing (a) standard recombinant methods, (b) synthetic techniques, orcombinations thereof. In some embodiments, the polynucleotides of thepresent invention can be cloned, amplified, or otherwise constructedfrom a monocot or dicot. Typical examples of monocots are corn, sorghum,barley, wheat, millet, rice, or turf grass. Typical dicots includesoybeans, safflower, sunflower, canola, alfalfa, potato, or cassaya.

[0035] Functional fragments included in the invention can be obtainedusing primers which selectively hybridize under stringent conditions.Primers are generally at least 12 bases in length and can be as high as200 bases, but will generally be from 15 to 75, or more likely from 15to 50 bases. Functional fragments can be identified using a variety oftechniques such as restriction analysis, Southern analysis, primerextension analysis, and DNA sequence analysis.

[0036] The present invention includes a plurality of polynucleotidesthat encode for the identical amino acid sequence. The degeneracy of thegenetic code allows for such “silent variations” which can be used, forexample, to selectively hybridize and detect allelic variants ofpolynucleotides of the present invention. Additionally, the presentinvention includes isolated nucleic acids comprising allelic variants.The term “allele” as used herein refers to a related nucleic acid of thesame gene.

[0037] Variants of nucleic acids included in the invention can beobtained, for example, by oligonucleotide-directed mutagenesis,linker-scanning mutagenesis, mutagenesis using the polymerase chainreaction, and the like. See, for example, pages 8.0.3-8.5.9 CurrentProtocols in Molecular Biology, Ausubel et al., Eds., Greene Publishingand Wiley-Interscience, New York (1995). Also, see generally, McPherson(ed.), DIRECTED MUTAGENESIS: A Practical Approach, (IRL Press, 1991).Thus, the present invention also encompasses DNA molecules comprisingnucleotide sequences that have substantial sequence similarity with theinventive sequences.

[0038] Variants included in the invention may contain individualsubstitutions, deletions or additions to the nucleic acid or polypeptidesequences which alter, add or delete a single amino acid or a smallpercentage of amino acids in the encoded sequence. A “conservativelymodified variant” is an alteration which results in the substitution ofan amino acid with a chemically similar amino acid. When the nucleicacid is prepared or altered synthetically, advantage can be taken ofknown codon preferences of the intended host.

[0039] With respect to particular nucleic acid sequences, conservativelymodified variants refers to those nucleic acids which encode identicalor conservatively modified variants of the amino acid sequences. Becauseof the degeneracy of the genetic code, a large number of functionallyidentical nucleic acids encode any given protein. For instance, thecodons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, atevery position where an alanine is specified by a codon, the codon canbe altered to any of the corresponding codons described without alteringthe encoded polypeptide. Such nucleic acid variations are “silentvariations” and represent one species of conservatively modifiedvariation. Every nucleic acid sequence herein that encodes a polypeptidealso, by reference to the genetic code, describes every possible silentvariation of the nucleic acid. One of ordinary skill will recognize thateach codon in a nucleic acid (except AUG, which is ordinarily the onlycodon for methionine; and UGG, which is ordinarily the only codon fortryptophan) can be modified to yield a functionally identical molecule.Accordingly, each silent variation of a nucleic acid which encodes apolypeptide of the present invention is implicit in each describedpolypeptide sequence and is within the scope of the claimed invention.

[0040] As to amino acid sequences, one of skill will recognize thatindividual substitutions, deletions or additions to a nucleic acid,peptide, polypeptide, or protein sequence which alters, adds or deletesa single amino acid or a small percentage of amino acids in the encodedsequence is a “conservatively modified variant” where the alterationresults in the substitution of an amino acid with a chemically similaramino acid. Thus, any number of amino acid residues selected from thegroup of integers consisting of from 1 to 15 can be so altered. Thus,for example, 1, 2, 3, 4, 5, 7, or 10 alterations can be made.Conservatively modified variants typically provide similar biologicalactivity as the unmodified polypeptide sequence from which they arederived. For example, substrate specificity, enzyme activity, orligand/receptor binding is generally at least 30%, 40%, 50%, 60%, 70%,80%, or 90% of the native protein for its native substrate. Conservativesubstitution tables providing functionally similar amino acids are wellknown in the art.

[0041] For example, the following six groups each contain amino acidsthat are conservative substitutions for one another:

[0042] 1) Alanine (A), Serine (S), Threonine (T);

[0043] 2) Aspartic acid (D), Glutamic acid (E);

[0044] 3) Asparagine (N), Glutamine (Q);

[0045] 4) Arginine (R), Lysine (K);

[0046] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

[0047] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0048] See also, Creighton (1984) Proteins W. H. Freeman and Company,other acceptable conservative substitution patterns known in the art mayalso be used, such as the scoring matrices of sequence comparisonprograms like the GCG package, BLAST, or CLUSTAL for example.

[0049] The claimed invention also includes “shufflents” produced bysequence shuffling of the inventive polynucleotides to obtain a desiredcharacteristic. Sequence shuffling is described in PCT publication No.96/19256. See also, Zhang, J. H., et al., Proc. Natl. Acad. Sci. USA94:4504-4509 (1997).

[0050] The present invention also includes the use of 5′ and/or 3′ UTRregions for modulation of translation of heterologous coding sequences.Positive sequence motifs include translational initiation consensussequences (Kozak, Nucleic Acids Res. 15:8125 (1987)) and the7-methylguanosine cap structure (Drummond et al., Nucleic Acids Res.13:7375 (1985)). Negative elements include stable intramolecular 5′ UTRstem-loop structures (Muesing et al., Cell 48:691 (1987)) and AUGsequences or short open reading frames preceded by an appropriate AUG inthe 5′ UTR (Kozak, supra, Rao et al., Mol. Cell. Biol. 8:284 (1988)).

[0051] Further, the polypeptide-encoding segments of the polynucleotidesof the present invention can be modified to alter codon usage. Alteredcodon usage can be employed to alter translational efficiency. Codonusage in the coding regions of the polynucleotides of the presentinvention can be analyzed statistically using commercially availablesoftware packages such as “Codon Preference” available from theUniversity of Wisconsin Genetics Computer Group (see Devereaux et al.,Nucleic Acids Res. 12:387-395 (1984)) or MacVector 4.1 (Eastman KodakCo., New Haven, Conn.).

[0052] For example, the inventive nucleic acids can be optimized forenhanced expression in plants of interest. See, for example, Perlak etal. (1991) Proc. Natl. Acad. Sci. USA 88:3324-3328; and Murray et al.(1989) Nucleic Acids Res. 17:477-498, the disclosure of which isincorporated herein by reference. In this manner, the polynucleotidescan be synthesized utilizing plant-preferred codons.

[0053] The present invention provides subsequences comprising isolatednucleic acids containing at least 20 contiguous bases of the claimedsequences. For example the isolated nucleic acid includes thosecomprising at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400,500, 600, 700 or 800 contiguous nucleotides of the claimed sequences.Subsequences of the isolated nucleic acid can be used to modulate ordetect gene expression by introducing into the subsequences compoundswhich bind, intercalate, cleave and/or crosslink to nucleic acids.

[0054] The nucleic acids of the claimed invention may convenientlycomprise a multi-cloning site comprising one or more endonucleaserestriction sites inserted into the nucleic acid to aid in isolation ofthe polynucleotide. Also, translatable sequences may be inserted to aidin the isolation of the translated polynucleotide of the presentinvention. For example, a hexa-histidine marker sequence, or a GSTfusion sequence, provides a convenient means to purify the proteins ofthe claimed invention.

[0055] A polynucleotide of the claimed invention can be attached to avector, adapter, promoter, transit peptide or linker for cloning and/orexpression of a polynucleotide of the present invention. Additionalsequences may be added to such cloning and/or expression sequences tooptimize their function in cloning and/or expression, to aid inisolation of the polynucleotide, or to improve the introduction of thepolynucleotide into a cell. Use of cloning vectors, expression vectors,adapters, and linkers is well known and extensively described in theart. For a description of such nucleic acids see, for example,Stratagene Cloning Systems, Catalogs 1995, 1996, 1997 (La Jolla,Calif.); and, Amersham Life Sciences, Inc, Catalog '97 (ArlingtonHeights, Ill.).

[0056] The isolated nucleic acid compositions of this invention, such asRNA, cDNA, genomic DNA, or a hybrid thereof, can be obtained from plantbiological sources using any number of cloning methodologies known tothose of skill in the art. In some embodiments, oligonucleotide probeswhich selectively hybridize, under stringent conditions, to thepolynucleotides of the present invention are used to identify thedesired sequence in a cDNA or genomic DNA library.

[0057] Exemplary total RNA and mRNA isolation protocols are described inPlant Molecular Biology: A Laboratory Manual, Clark, Ed.,Springer-Verlag, Berlin (1997); and, Current Protocols in MolecularBiology, Ausubel et al., Eds., Greene Publishing and Wiley-Interscience,New York (1995). Total RNA and mRNA isolation kits are commerciallyavailable from vendors such as Stratagene (La Jolla, Calif.), Clonetech(Palo Alto, Calif.), Pharmacia (Piscataway, N.J.), and 5′-3′ (Paoli,Pa.). See also, U.S. Pat. Nos. 5,614,391; and, 5,459,253.

[0058] Typical cDNA synthesis protocols are well known to the skilledartisan and are described in such standard references as: PlantMolecular Biology: A Laboratory Manual, Clark, Ed., Springer-Verlag,Berlin (1997); and, Current Protocols in Molecular Biology, Ausubel etal., Eds., Greene Publishing and Wiley-Interscience, New York (1995).cDNA synthesis kits are available from a variety of commercial vendorssuch as Stratagene or Pharmacia.

[0059] An exemplary method of constructing a greater than 95% purefull-length cDNA library is described by Carninci et al., Genomics37:327-336 (1996). Other methods for producing full-length libraries areknown in the art. See, e.g., Edery et al., Mol. Cell Biol.15(6):3363-3371 (1995); and PCT Application WO 96/34981.

[0060] It is often convenient to normalize a cDNA library to create alibrary in which each clone is more equally represented. A number ofapproaches to normalize cDNA libraries are known in the art.Construction of normalized libraries is described in Ko, Nucl. Acids.Res. 18(19):5705-5711 (1990); Patanjali et al., Proc. Natl. Acad. U.S.A.88:1943-1947 (1991); U.S. Pat. Nos. 5,482,685 and 5,637,685; and Soareset al., Proc. Natl. Acad. Sci. USA 91:9228-9232 (1994).

[0061] Subtracted cDNA libraries are another means to increase theproportion of less abundant CDNA species. See, Foote et al. in, PlantMolecular Biology: A Laboratory Manual, Clark, Ed., Springer-Verlag,Berlin (1997); Kho and Zarbl, Technique 3(2):58-63 (1991); Sive and St.John, Nucl. Acids Res. 16(22):10937 (1988); Current Protocols inMolecular Biology, Ausubel et al., Eds., Greene Publishing andWiley-Interscience, New York (1995); and, Swaroop et al., Nucl. AcidsRes. 19(8):1954 (1991). cDNA subtraction kits are commerciallyavailable. See, e.g., PCR-Select (Clontech).

[0062] To construct genomic libraries, large segments of genomic DNA aregenerated by random fragmentation. Examples of appropriate molecularbiological techniques and instructions are found in Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring HarborLaboratory, Vols. 1-3 (1989), Methods in Enzymology, Vol. 152: Guide toMolecular Cloning Techniques, Berger and Kimmel, Eds., San Diego:Academic Press, Inc. (1987), Current Protocols in Molecular Biology,Ausubel et al., Eds., Greene Publishing and Wiley-Interscience, New York(1995); Plant Molecular Biology: A Laboratory Manual, Clark, Ed.,Springer-Verlag, Berlin (1997). Kits for construction of genomiclibraries are also commercially available.

[0063] The cDNA or genomic library can be screened using a probe basedupon the sequence of a nucleic acid of the present invention such asthose disclosed herein. Probes may be used to hybridize with genomic DNAor cDNA sequences to isolate homologous polynucleotides in the same ordifferent plant species. Those of skill in the art will appreciate thatvarious degrees of stringency of hybridization can be employed in theassay; and either the hybridization or the wash medium can be stringent.The degree of stringency can be controlled by temperature, ionicstrength, pH and the presence of a partially denaturing solvent such asformamide.

[0064] Typically, stringent hybridization conditions will be those inwhich the salt concentration is less than about 1.5 M Na ion, typicallyabout 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to8.3 and the temperature is at least about 30° C. for short probes (e.g.,10 to 50 nucleotides) and at least about 60° C. for long probes (e.g.,greater than 50 nucleotides). Stringent conditions may also be achievedwith the addition of destabilizing agents such as formamide.

[0065] Exemplary low stringency conditions include hybridization with abuffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecylsulfate) at 37° C., and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 Mtrisodium citrate) at 50° C. Exemplary moderate stringency conditionsinclude hybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37°C., and a wash in 0.5× to 1× SSC at 55° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a wash in 0.1×SSC at 60° C. Typically the time ofhybridization is from 4 to 16 hours.

[0066] An extensive guide to the hybridization of nucleic acids is foundin Tijssen, Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2“Overview of principles of hybridization and the strategy of nucleicacid probe assays”, Elsevier, N.Y. (1993); and Current Protocols inMolecular Biology, Chapter 2, Ausubel et al., Eds., Greene Publishingand Wiley-Interscience, New York (1995). Often, cDNA libraries will benormalized to increase the representation of relatively rare cDNAs.

[0067] The nucleic acids of the invention can be amplified from nucleicacid samples using amplification techniques. For instance, polymerasechain reaction (PCR) technology can be used to amplify the sequences ofpolynucleotides of the present invention and related polynucleotidesdirectly from genomic DNA or cDNA libraries. PCR and other in vitroamplification methods may also be useful, for example, to clone nucleicacid sequences that code for proteins to be expressed, to make nucleicacids to use as probes for detecting the presence of the desired mRNA insamples, for nucleic acid sequencing, or for other purposes.

[0068] Examples of techniques useful for in vitro amplification methodsare found in Berger, Sambrook, and Ausubel, as well as Mullis et al.,U.S. Pat. No. 4,683,202 (1987); and, PCR Protocols A Guide to Methodsand Applications, Innis et al., Eds., Academic Press Inc., San Diego,Calif. (1990). Commercially available kits for genomic PCR amplificationare known in the art. See, e.g., Advantage-GC Genomic PCR Kit(Clontech). The T4 gene 32 protein (Boehringer Mannheim) can be used toimprove yield of long PCR products. PCR-based screening methods havealso been described. Wilfinger et al. describe a PCR-based method inwhich the longest cDNA is identified in the first step so thatincomplete clones can be eliminated from study. BioTechniques,22(3):481-486 (1997).

[0069] In one aspect of the invention, nucleic acids can be amplifiedfrom a plant nucleic acid library. The nucleic acid library may be acDNA library, a genomic library, or a library generally constructed fromnuclear transcripts at any stage of intron processing. Libraries can bemade from a variety of plant tissues such as ears, seedlings, leaves,stalks, roots, pollen, or seeds. Good results have been obtained usingtissues such as night-harvested earshoot with husk at stage V-12 fromcorn line B73, corn night-harvested leaf tissue at stage V8-V10 fromline B73, corn anther tissue at prophase I from line B73, 4 DAPcoenocytic embryo sacs from corn line B73, 67 day old corn cob from cornline L, and corn BMS suspension cells treated with chemicals related tophosphatases.

[0070] Alternatively, the sequences of the invention can be used toisolate corresponding sequences in other organisms, particularly otherplants, more particularly, other monocots. In this manner, methods suchas PCR, hybridization, and the like can be used to identify suchsequences having substantial sequence similarity to the sequences of theinvention. See, for example, Sambrook et al. (1989) Molecular Cloning: ALaboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press,Plainview, N.Y.) and Innis et al. (1990), PCR Protocols: A Guide toMethods and Applications (Academic Press, New York). Coding sequencesisolated based on their sequence identity to the entire inventive codingsequences set forth herein or to fragments thereof are encompassed bythe present invention.

[0071] The isolated nucleic acids of the present invention can also beprepared by direct chemical synthesis by methods such as thephosphotriester method of Narang et al., Meth. Enzymol. 68:90-99 (1979);the phosphodiester method of Brown et al., Meth. Enzymol. 68:109-151(1979); the diethylphosphoramidite method of Beaucage et al., Tetra.Lett. 22:1859-1862 (1981); the solid phase phosphoramidite triestermethod described by Beaucage and Caruthers, Tetra. Lett.22(20):1859-1862 (1981), e.g., using an automated synthesizer, e.g., asdescribed in Needham-VanDevanter et al., Nucleic Acids Res. 12:6159-6168(1984); and, the solid support method of U.S. Pat. No. 4,458,066.Chemical synthesis generally produces a single stranded oligonucleotide.This may be converted into double stranded DNA by hybridization with acomplementary sequence, or by polymerization with a DNA polymerase usingthe single strand as a template. One of skill will recognize that whilechemical synthesis of DNA is limited to sequences of about 100 bases,longer sequences may be obtained by the ligation of shorter sequences.

[0072] The nucleic acids of the claimed invention include thoseamplified using the following primer pairs: SEQ ID NO: 15 paired withSEQ ID NO: 16,17, 22 or 27.

Expression Cassettes

[0073] In another embodiment expression cassettes comprising isolatednucleic acids of the present invention are provided. An expressioncassette will typically comprise a polynucleotide of the presentinvention operably linked to transcriptional initiation regulatorysequences which will direct the transcription of the polynucleotide inthe intended host cell, such as tissues of a transformed plant.

[0074] The construction of such expression cassettes which can beemployed in conjunction with the present invention is well known tothose of skill in the art in light of the present disclosure. See, e.g.,Sambrook et al.; Molecular Cloning: A Laboratory Manual; Cold SpringHarbor, N.Y.; (1989); Gelvin et al.; Plant Molecular Biology Manual(1990); Plant Biotechnology: Commercial Prospects and Problems, eds.Prakash et al.; Oxford & IBH Publishing Co.; New Delhi, India; (1993);and Heslot et al.; Molecular Biology and Genetic Engineering of Yeasts;CRC Press, Inc., USA; (1992); each incorporated herein in its entiretyby reference.

[0075] For example, plant expression vectors may include (1) a clonedplant gene under the transcriptional control of 5′ and 3′ regulatorysequences and (2) a dominant selectable marker. Such plant expressionvectors may also contain, if desired, a promoter regulatory region(e.g., one conferring inducible, constitutive, environmentally- ordevelopmentally-regulated, or cell- or tissue-specific/selectiveexpression), a transcription initiation start site, a ribosome bindingsite, an RNA processing signal, a transcription termination site, and/ora polyadenylation signal.

[0076] Constitutive, tissue-preferred or inducible promoters can beemployed. Examples of constitutive promoters include the cauliflowermosaic virus (CaMV) 35S transcription initiation region, the 1′- or2′-promoter derived from T-DNA of Agrobacterium tumefaciens, the actinpromoter, the ubiquitin promoter, the histone H2B promoter (Nakayama etal., 1992, FEBS Lett 30:167-170), the Smas promoter, the cinnamylalcohol dehydrogenase promoter (U.S. Pat. No. 5,683,439), the Nospromoter, the pEmu promoter, the rubisco promoter, the GRP1-8 promoter,and other transcription initiation regions from various plant genesknown in the art.

[0077] Examples of inducible promoters are the Adh1 promoter which isinducible by hypoxia or cold stress, the Hsp70 promoter which isinducible by heat stress, the PPDK promoter which is inducible by light,the In2 promoter which is safener induced, the ERE promoter which isestrogen induced and the pepcarboxylase promoter which is light induced.

[0078] Examples of promoters under developmental control includepromoters that initiate transcription preferentially in certain tissues,such as leaves, roots, fruit, pollen, seeds, or flowers. An exemplarypromoter is the anther specific promoter 5126 (U.S. Pat. Nos. 5,689,049and 5,689,051). Examples of seed-preferred promoters include, but arenot limited to, 27 kD gamma zein promoter and waxy promoter, (Boronat,A., et al., Plant Sci. 47:95-102 (1986); Reina, M., et al., NucleicAcids Res. 18(21):6426 (1990); Kloesgen, R. B., et al., Mol. Gen. Genet.203:237-244 (1986)), as well as the globulin 1, oleosin and thephaseolin promoters. The disclosures each of these are incorporatedherein by reference in their entirety.

[0079] The barley or maize Nuc1 promoter, the maize Cim1 promoter or themaize LTP2 promoter can be used to preferentially express in thenucellus. See, for example WO 00/11177, the disclosure of which isincorporated herein by reference.

[0080] Either heterologous or non-heterologous (i.e., endogenous)promoters can be employed to direct expression of the nucleic acids ofthe present invention. These promoters can also be used, for example, inexpression cassettes to drive expression of sense nucleic acids orantisense nucleic acids to reduce, increase, or alter concentrationand/or composition of the proteins of the present invention in a desiredtissue.

[0081] If polypeptide expression is desired, it is generally desirableto include a polyadenylation region at the 3′-end of a polynucleotidecoding region. The polyadenylation region can be derived from thenatural gene, from a variety of other plant genes, or from T-DNA. The 3′end sequence to be added can be derived from, for example, the nopalinesynthase or octopine synthase genes, or alternatively from another plantgene, or less preferably from any other eukaryotic gene.

[0082] An intron sequence can be added to the 5′ untranslated region orthe coding sequence of the partial coding sequence to increase theamount of the mature message that accumulates. See for example Buchmanand Berg, Mol. Cell Biol. 8:4395-4405 (1988); Callis et al., Genes Dev.1:1183-1200 (1987). Use of maize introns Adh1-S intron 1, 2, and 6, theBronze-1 intron are known in the art. See generally, The Maize Handbook,Chapter 116, Freeling and Walbot, Eds., Springer, New York (1994).

[0083] The vector comprising the sequences from a polynucleotide of thepresent invention will typically comprise a marker gene which confers aselectable phenotype on plant cells. Usually, the selectable marker geneencodes antibiotic or herbicide resistance. Suitable genes include thosecoding for resistance to the antibiotics spectinomycin and streptomycin(e.g., the aada gene), the streptomycin phosphotransferase (SPT) genecoding for streptomycin resistance, the neomycin phosphotransferase(NPTII) gene encoding kanamycin or geneticin resistance, the hygromycinphosphotransferase (HPT) gene coding for hygromycin resistance.

[0084] Suitable genes coding for resistance to herbicides include thosewhich act to inhibit the action of acetolactate synthase (ALS), inparticular the sulfonylurea-type herbicides (e.g., the acetolactatesynthase (ALS) gene containing mutations leading to such resistance inparticular the S4 and/or Hra mutations), those which act to inhibitaction of glutamine synthase, such as phosphinothricin or basta (e.g.,the bar gene), or other such genes known in the art. The bar geneencodes resistance to the herbicide basta and the ALS gene encodesresistance to the herbicide chlorsulfuron.

[0085] Typical vectors useful for expression of genes in higher plantsare well known in the art and include vectors derived from thetumor-inducing (Ti) plasmid of Agrobacterium tumefaciens described byRogers et al., Meth. In Enzymol. 153:253-277 (1987). Exemplary A.tumefaciens vectors useful herein are plasmids pKYLX6 and pKYLX7 ofSchardl et al., Gene 61:1-11 (1987) and Berger et al., Proc. Natl. Acad.Sci. USA 86:8402-8406 (1989). Another useful vector herein is plasmidpBI101.2 that is available from Clontech Laboratories, Inc. (Palo Alto,Calif.).

[0086] A variety of plant viruses that can be employed as vectors areknown in the art and include cauliflower mosaic virus (CaMV),geminivirus, brome mosaic virus, and tobacco mosaic virus.

[0087] A polynucleotide of the claimed invention can be expressed ineither sense or anti-sense orientation as desired. In plant cells, ithas been shown that antisense RNA inhibits gene expression by preventingthe accumulation of mRNA which encodes the enzyme of interest, see,e.g., Sheehy et al., Proc. Natl. Acad. Sci. USA 85:8805-8809 (1988); andHiatt et al., U.S. Pat. No. 4,801,340.

[0088] Another method of suppression is sense suppression. Introductionof nucleic acid configured in the sense orientation has been shown to bean effective means by which to block the transcription of target genes.For an example of the use of this method to modulate expression ofendogenous genes see, Napoli et al., The Plant Cell 2:279-289 (1990) andU.S. Pat. No. 5,034,323. Recent work has shown suppression with the useof double stranded RNA. Such work is described in Tabara et al., Science282:5388:430-431 (1998). Hairpin approaches of gene suppression aredisclosed in WO 98/53083 and WO 99/53050.

[0089] Catalytic RNA molecules or ribozymes can also be used to inhibitexpression of plant genes. The inclusion of ribozyme sequences withinantisense RNAs confers RNA-cleaving activity upon them, therebyincreasing the activity of the constructs. The design and use of targetRNA-specific ribozymes is described in Haseloff et al., Nature334:585-591 (1988).

[0090] A variety of cross-linking agents, alkylating agents and radicalgenerating species as pendant groups on polynucleotides of the presentinvention can be used to bind, label, detect, and/or cleave nucleicacids. For example, Vlassov, V. V., et al., Nucleic Acids Res (1986)14:4065-4076, describe covalent bonding of a single-stranded DNAfragment with alkylating derivatives of nucleotides complementary totarget sequences. A report of similar work by the same group is that byKnorre, D. G., et al., Biochimie (1985) 67:785-789. Iverson and Dervanalso showed sequence-specific cleavage of single-stranded DNA mediatedby incorporation of a modified nucleotide which was capable ofactivating cleavage (J. Am. Chem. Soc. (1987) 109:1241-1243). Meyer, R.B., et al., J. Am. Chem. Soc. (1989) 111:8517-8519, effect covalentcrosslinking to a target nucleotide using an alkylating agentcomplementary to the single-stranded target nucleotide sequence. Aphotoactivated crosslinking to single-stranded oligonucleotides mediatedby psoralen was disclosed by Lee, B. L., et al., Biochemistry (1988)27:3197-3203. Use of crosslinking in triple-helix forming probes wasalso disclosed by Home et al., J. Am. Chem. Soc. (1990) 112:2435-2437.Use of N4, N4-ethanocytosine as an alkylating agent to crosslink tosingle-stranded oligonucleotides has also been described by Webb andMafteucci, J. Am. Chem. Soc. (1986) 108:2764-2765; Nucleic Acids Res(1986) 14:7661-7674; Feteritz et al., J. Am. Chem. Soc. 113:4000 (1991).Various compounds to bind, detect, label, and/or cleave nucleic acidsare known in the art. See, for example, U.S. Pat. Nos. 5,543,507;5,672,593; 5,484,908; 5,256,648; and 5,681941.

Gene or Trait Stacking

[0091] In certain embodiments the nucleic acid sequences of the presentinvention can be stacked with any combination of polynucleotidesequences of interest in order to create plants with a desiredphenotype. For example, the polynucleotides of the present invention maybe stacked with any other polynucleotides of the present invention, suchas any combination of ITPK-2, ITPK-3, ITPK-4, ITPK-5, ITPK-6, and ITPK-7(SEQ ID NOS: 1, 3, 5, 7, 9, 11 and 13), or with other genes implicatedin phytic acid metabolic pathways such as phytase; Lpal, Lpa2 (see U.S.Pat. Nos. 5,689,054 and 6,111,168); myo-inositol 1-phosphate synthase (M1PS), inositol polyphosphate kinase (IPPK), and myo-inositolmonophophatase (IMP) (see WO 99/05298 and U.S. Application Serial No.10/042,465 filed Jan. 9, 2002) and the like, the disclosures of whichare herein incorporated by reference. The combinations generated canalso include multiple copies of any one of the polynucleotides ofinterest. The polynucleotides of the present invention can also bestacked with any other gene or combination of genes to produce plantswith a variety of desired trait combinations including but not limitedto traits desirable for animal feed such as high oil genes (e.g., U.S.Pat. No. 6,232,529); balanced amino acids (e.g. hordothionins (U.S. Pat.Nos. 5,990,389; 5,885,801; 5,885,802; and 5,703,409); barley high lysine(Williamson et al. (1987) Eur. J. Biochem. 165:99-106; and WO 98/20122);and high methionine proteins (Pedersen et al. (1986) J. Biol. Chem.261:6279; Kirihara et al. (1988) Gene 71:359; and Musumura et al. (1989)Plant Mol. Biol. 12: 123)); increased digestibility (e.g., modifiedstorage proteins (U.S. Application Serial No. 10/053,410, filed Nov. 7,2001); and thioredoxins (U.S. Application Serial No. 10/005,429, filedDec. 3, 2001)), the disclosures of which are herein incorporated byreference. The polynucleotides of the present invention can also bestacked with traits desirable for insect, disease or herbicideresistance (e.g., Bacillus thuringiensis toxic proteins (U.S. Pat. Nos.5,366,892; 5,747,450; 5,737,514; 5723,756; 5,593,881; Geiser et al(1986) Gene 48:109); lectins (Van Damme et al. (1994) Plant Mol. Biol.24:825); fumonisin detoxification genes (U.S. Pat. No. 5,792,931);avirulence and disease resistance genes (Jones et al. (1994) Science266:789; Martin et al. (1993) Science 262:1432; Mindrinos et al. (1994)Cell 78:1089); acetolactate synthase (ALS) mutants that lead toherbicide resistance such as the S4 and/or Hra mutations; inhibitors ofglutamine synthase such as phosphinothricin or basta (e.g., bar gene);and glyphosate resistance (EPSPS gene)); and traits desirable forprocessing or process products such as high oil (e.g., U.S. Pat. No.6,232,529); modified oils (e.g., fafty acid desaturase genes (U.S. Pat.No. 5,952,544; WO 94/11516)); modified starches (e.g., ADPGpyrophosphorylases (AGPase), starch synthases (SS), starch branchingenzymes (SBE) and starch debranching enzymes (SDBE)); and polymers orbioplastics (e.g., U.S. Pat. No. 5,602,321; beta-ketothiolase,polyhydroxybutyrate synthase, and acetoacetyl-CoA reductase (Schubert etal. (1988) J. Bacteriol. 170:5837-5847) facilitate expression ofpolyhydroxyalkanoates (PHAs)), the disclosures of which are hereinincorporated by reference. One could also combine the polynucleotides ofthe present invention with polynucleotides providing agronomic traitssuch as male sterility (e.g., see U.S. Pat. No. 5,583,210), stalkstrength, flowering time, or transformation technology traits such ascell cycle regulation or gene targeting (e.g. WO 99/61619; WO 00/17364;WO 99/25821), the disclosures of which are herein incorporated byreference.

[0092] These stacked combinations can be created by any method includingbut not limited to cross breeding plants by any conventional or TopCrossmethodology, or genetic transformation. If the traits are stacked bygenetically transforming the plants, the polynucleotide sequences ofinterest can be combined at any time and in any order. For example, atransgenic plant comprising one or more desired traits can be used asthe target to introduce further traits by subsequent transformation. Thetraits can be introduced simultaneously in a co-transformation protocolwith the polynucleotides of interest provided by any combination oftransformation cassettes. For example, if two sequences will beintroduced, the two sequences can be contained in separatetransformation cassettes (trans) or contained on the same transformationcassette (cis). Expression of the sequences can be driven by the samepromoter or by different promoters. In certain cases, it may bedesirable to introduce a transformation cassette that will suppress theexpression of the polynucleotide of interest. This may be combine withany combination of other suppression cassettes or overexpressioncassettes to generate the desired combination of traits in the plant.

Proteins

[0093] ITPK proteins are involved in the phosphorylation of appropriateinositol phosphate substrates in inositol phosphate metabolism. Theseenzymes have a broader substrate specificity than earlier suspected andcan phosphorylate various species of IP₃ and IP₄, using ATP as thephosphate donor. The proteins of the present invention show homology toknown ITPK sequences, with the sequence similarity distributed acrossthe entire sequence. It is expected that modulation of the expression ofthese proteins of the present invention will provide methods to improvethe quality of animal feed by reducing the level of phytate and/orincreasing the level of bioavailable phosphorous. Reducing phytatelevels could also result in less environment-polluting phosphorous inthe waste of non-ruminant animals.

[0094] Proteins of the present invention include proteins having thedisclosed sequences as well proteins coded by the disclosedpolynucleotides. In addition, proteins of the present invention includeproteins derived from the native protein by deletion, addition orsubstitution of one or more amino acids at one or more sites in thenative protein. Such variants may result from, for example, geneticpolymorphism or from human manipulation. Methods for such manipulationsare generally known in the art.

[0095] For example, amino acid sequence variants of the polypeptide canbe prepared by mutations in the cloned DNA sequence encoding the nativeprotein of interest. Methods for mutagenesis and nucleotide sequencealterations are well known in the art. See, for example, Walker andGaastra, eds. (1983) Techniques in Molecular Biology (MacMillanPublishing Company, New York); Kunkel (1985) Proc. Natl. Acad. Sci. USA82:488-492; Kunkel et al. (1987) Methods Enzymol. 154:367-382; Sambrooket al. (1989) Molecular Cloning: A Laboratory Manual (Cold SpringHarbor, N.Y.); U.S. Pat. No. 4,873,192; and the references citedtherein; herein incorporated by reference. Guidance as to appropriateamino acid substitutions that do not affect biological activity of theprotein of interest may be found in the model of Dayhoff et al. (1978)Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found.,Washington, D.C.), herein incorporated by reference. Conservativesubstitutions, such as exchanging one amino acid with another havingsimilar properties, may be preferred.

[0096] In constructing variants of the proteins of interest,modifications to the nucleotide sequences encoding the variants cangenerally be made such that variants continue to possess the desiredactivity.

[0097] The isolated proteins of the present invention include apolypeptide comprising at least 25 contiguous amino acids encoded by anyone of the nucleic acids of the present invention, or polypeptides thatare conservatively modified variants thereof. The proteins of thepresent invention or variants thereof can comprise any number ofcontiguous amino acid residues from a polypeptide of the presentinvention, wherein that number is selected from the group of integersconsisting of from 25 to the number of residues in a full-lengthpolypeptide of the present invention. Optionally, this subsequence ofcontiguous amino acids is at least 25, 30, 40, 50, 60, 70, 80, 90,100,150, 200, 250, 300, 350, 400, 450, or 500 amino acids in length.

[0098] The present invention includes catalytically active polypeptides(i.e., enzymes). Catalytically active polypeptides will generally have aspecific activity of at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, or 95% that of the native (non-synthetic), endogenous polypeptide.Further, the substrate specificity (k_(cat)/K_(m)) is optionallysubstantially similar to the native (non-synthetic), endogenouspolypeptide. Typically, the K_(m) will be at least about 30%, 40%, 50%,60%, 70%, 80%, 90%, or 95% that of the native (non-synthetic),endogenous polypeptide. Methods of assaying and quantifying measures ofenzymatic activity and substrate specificity (k_(cat)/K_(m)), are wellknown to those of skill in the art. See, e.g., Segel, BiochemicalCalculations, 2^(nd) ed., John Wiley and Sons, New York (1976).

[0099] The present invention includes modifications that can be made toan inventive protein. In particular, it may be desirable to diminish theactivity of the gene. Other modifications may be made to facilitate thecloning, expression, or incorporation of the targeting molecule into afusion protein. Such modifications are well known to those of skill inthe art and include, for example, a methionine added at the aminoterminus to provide an initiation site, or additional amino acids (e.g.,poly His) placed on either terminus to create conveniently locatedrestriction sites or termination codons or purification sequences.

[0100] Using the nucleic acids of the present invention, one may expressa protein of the present invention in recombinantly engineered cellssuch as bacteria, yeast, insect, mammalian, or plant cells. The cellsproduce the protein in a non-natural condition (e.g., in quantity,composition, location, and/or time), because they have been geneticallyaltered through human intervention to do so.

[0101] Typically, an intermediate host cell may be used in the practiceof this invention to increase the copy number of the cloning vector.With an increased copy number, the vector containing the gene ofinterest can be isolated in significant quantities for introduction intothe desired plant cells.

[0102] Host cells that can be used in the practice of this inventioninclude prokaryotes and eukaryotes. Prokaryotes include bacterial hostssuch as Eschericia coli, Salmonella typhimurium, and Serratiamarcescens. Eukaryotic hosts such as yeast, insect cells or filamentousfungi may also be used in this invention.

[0103] Commonly used prokaryotic control sequences include such commonlyused promoters as the beta lactamase (penicillinase) and lactose (lac)promoter systems (Chang et al., Nature 198:1056 (1977)), the tryptophan(trp) promoter system (Goeddel et al., Nucleic Acids Res. 8:4057 (1980))and the lambda derived P L promoter and N-gene ribosome binding site(Shimatake et al., Nature 292:128 (1981)). The inclusion of selectionmarkers in DNA vectors transfected in E. coli is also useful. Examplesof such markers include genes specifying resistance to ampicillin,tetracycline, or chloramphenicol.

[0104] The vector is selected to allow introduction into the appropriatehost cell. Bacterial vectors are typically of plasmid or phage origin.Expression systems for expressing a protein of the present invention areavailable using Bacillus sp. and Salmonella (Palva et al., Gene22:229-235 (1983); Mosbach et al., Nature 302:543-545 (1983)).

[0105] Synthesis of heterologous proteins in yeast is well known. SeeSherman, F., et al., Methods in Yeast Genetics, Cold Spring HarborLaboratory (1982). Two widely utilized yeast for production ofeukaryotic proteins are Saccharomyces cerevisiae and Pichia pastoris.Vectors, strains, and protocols for expression in Saccharomyces andPichia are known in the art and available from commercial suppliers(e.g., Invitrogen). Suitable vectors usually have expression controlsequences, such as promoters, including 3-phosphoglycerate kinase oralcohol oxidase, and an origin of replication, termination sequences andthe like as desired.

[0106] The baculovirus expression system (BES) is a eukaryotic,helper-independent expression system which has been used to expresshundreds of foreign genes (Luckow, V., Ch. 4 “Cloning and Expression ofHeterologous Genes in Insect Cells with Baculovirus Vectors” inRecombinant DNA Technology and Applications, A. Prokop et al., Eds.McGraw-Hill, Inc. (1991); Luckow, V., Ch. 10 “Insect ExpressionTechnology” in Principles & Practice of Protein Engineering, J. L.Cleland and C. S. Craig, Eds. John Wiley & Sons, (1994)).

[0107] Recombinant baculoviruses are generated by inserting theparticular gene- or genes-of-interest into the baculovirus genome usingestablished protocols with vectors and reagents from commercialsuppliers (e.g., Invitrogen, Life Technologies Incorporated). Commercialvectors are readily available with various promoters, such as polyhedrinand p10, optional signal sequences for protein secretion, or affinitytags, such as 6×histidine. These recombinant viruses are grown,maintained and propagated in commercially available cell lines derivedfrom several insect species including Spodoptera frugiperda andTrichoplusia ni. The insect cells can be cultured using well-establishedprotocols in a variety of different media, for example, with and withoutbovine serum supplementation. The cultured cells are infected with therecombinant viruses and the gene-of-interest polypeptide is expressed.Proteins expressed with the baculovirus system have been extensivelycharacterized and, in many cases, their post-translational modificationssuch as phosphorylation, acylation, etc., are identical to the nativelyexpressed protein.

[0108] A protein of the present invention, once expressed, can beisolated from cells by lysing the cells and applying standard proteinisolation techniques to the lysates. The monitoring of the purificationprocess can be accomplished by using Western blot techniques orradioimmunoassay or other standard immunoassay techniques. Expressioncassettes are also available which direct the expressed protein to besecreted from the cell into the media. In these cases, the expressedprotein can be purified from the cell growth media using standardprotein purification techniques.

[0109] The proteins of the present invention can also be constructedusing non-cellular synthetic methods. Solid phase synthesis of proteinsof less than about 50 amino acids in length may be accomplished byattaching the C-terminal amino acid of the sequence to an insolublesupport followed by sequential addition of the remaining amino acids inthe sequence. Techniques for solid phase synthesis are described byBarany and Merrifield, Solid-Phase Peptide Synthesis, pp. 3-284 in ThePeptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods inPeptide Synthesis, Part A.; Merrifield et al., J. Am. Chem. Soc.85:2149-2156 (1963), and Stewart et al., Solid Phase Peptide Synthesis,2nd ed., Pierce Chem. Co., Rockford, Ill. (1984). Proteins of greaterlength may be synthesized by condensation of the amino and carboxytermini of shorter fragments. Methods of forming peptide bonds byactivation of a carboxy terminal end (e.g., by the use of the couplingreagent N,N′-dicyclohexylcarbodiimide)) are known to those of skill.

[0110] The proteins of this invention, recombinant or synthetic, may bepurified to substantial purity by standard techniques well known in theart, including detergent solubilization, selective precipitation withsuch substances as ammonium sulfate, column chromatography,immunopurification methods, and others. See, for instance, R. Scopes,Protein Purification: Principles and Practice, Springer-Verlag: New York(1982); Deutscher, Guide to Protein Purification, Academic Press (1990).For example, antibodies may be raised to the proteins as describedherein. Purification from E. coli can be achieved following proceduresdescribed in U.S. Pat. No. 4,511,503. Detection of the expressed proteinis achieved by methods known in the art and include, for example,radioimmunoassays, Western blotting techniques or immunoprecipitation.

[0111] The present invention further provides a method for modulating(i.e., increasing or decreasing) the concentration or composition of thepolypeptides of the claimed invention in a plant or part thereof.Modulation can be effected by increasing or decreasing the concentrationand/or the composition (i.e., the ratio of the polypeptides of theclaimed invention) in a plant.

[0112] The method comprises transforming a plant cell with an expressioncassette comprising a polynucleotide of the present invention to obtaina transformed plant cell, growing the transformed plant cell underconditions allowing expression of the polynucleotide in the plant cellin an amount sufficient to modulate concentration and/or composition inthe plant cell.

[0113] In some embodiments, the content and/or composition ofpolypeptides of the present invention in a plant may be modulated byaltering, in vivo or in vitro, the promoter of a non-isolated gene ofthe present invention to up- or down-regulate gene expression. In someembodiments, the coding regions of native genes of the present inventioncan be altered via substitution, addition, insertion, or deletion todecrease activity of the encoded enzyme. See, e.g., Kmiec, U.S. Pat. No.5,565,350; Zarling et al., PCT/US93/03868. One method of down-regulationof the protein involves using PEST sequences that provide a target fordegradation of the protein.

[0114] In some embodiments, an isolated nucleic acid (e.g., a vector)comprising a promoter sequence is transfected into a plant cell.Subsequently, a plant cell comprising the promoter operably linked to apolynucleotide of the present invention is selected for by means knownto those of skill in the art such as, but not limited to, Southern blot,DNA sequencing, or PCR analysis using primers specific to the promoterand to the gene and detecting amplicons produced therefrom. A plant orplant part altered or modified by the foregoing embodiments is grownunder plant forming conditions for a time sufficient to modulate theconcentration and/or composition of polypeptides of the presentinvention in the plant. Plant forming conditions are well known in theart.

[0115] In general, content of the polypeptide is increased or decreasedby at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% relativeto a native control plant, plant part, or cell lacking theaforementioned expression cassette. Modulation in the present inventionmay occur during and/or subsequent to growth of the plant to the desiredstage of development. Modulating nucleic acid expression temporallyand/or in particular tissues can be controlled by employing theappropriate promoter operably linked to a polynucleotide of the presentinvention in, for example, sense or antisense orientation as discussedin greater detail, supra. Induction of expression of a polynucleotide ofthe present invention can also be controlled by exogenous administrationof an effective amount of inducing compound. Inducible promoters andinducing compounds which activate expression from these promoters arewell known in the art. In certain embodiments, the polypeptides of thepresent invention are modulated in monocots or dicots, for examplemaize, soybeans, sunflower, safflower, sorghum, canola, wheat, alfalfa,rice, barley and millet.

[0116] Means of detecting the proteins of the present invention are notcritical aspects of the present invention. The proteins can be detectedand/or quantified using any of a number of well-recognized immunologicalbinding assays (see, e.g., U.S. Pat. Nos. 4,366,241; 4,376,110;4,517,288; and 4,837,168). Fora review of the general immunoassays, seealso Methods in Cell Biology, Vol. 37: Antibodies in Cell Biology, Asai,Ed., Academic Press, Inc. New York (1993); Basic and Clinical Immunology7th Edition, Stites & Terr, Eds. (1991). Moreover, the immunoassays ofthe present invention can be performed in any of several configurations,e.g., those reviewed in Enzyme Immunoassay, Maggio, Ed., CRC Press, BocaRaton, Fla. (1980); Tijan, Practice and Theory of Enzyme Immunoassays,Laboratory Techniques in Biochemistry and Molecular Biology, ElsevierScience Publishers B. V., Amsterdam (1985); Harlow and Lane, supra;Immunoassay: A Practical Guide, Chan, Ed., Academic Press, Orlando, Fla.(1987); Principles and Practice of Immunoassays, Price and Newman Eds.,Stockton Press, NY (1991); and Non-isotopic Immunoassays, Ngo, Ed.,Plenum Press, NY (1988).

[0117] Typical methods include Western blot (immunoblot) analysis,analytic biochemical methods such as electrophoresis, capillaryelectrophoresis, high performance liquid chromatography (HPLC), thinlayer chromatography (TLC), hyperdiffusion chromatography, and the like,and various immunological methods such as fluid or gel precipitinreactions, immunodiffusion (single or double), immunoelectrophoresis,radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs),immunofluorescent assays, and the like.

[0118] Non-radioactive labels are often attached by indirect means.Generally, a ligand molecule (e.g., biotin) is covalently bound to themolecule. The ligand then binds to an anti-ligand molecule (e.g.,streptavidin) which is either inherently detectable or covalently boundto a signal system, such as a detectable enzyme, a fluorescent compound,or a chemiluminescent compound. A number of ligands and anti-ligands canbe used. Where a ligand has a natural anti-ligand, for example, biotin,thyroxine, and cortisol, it can be used in conjunction with the labeled,naturally occurring anti-ligands. Alternatively, any haptenic orantigenic compound can be used in combination with an antibody.

[0119] The molecules can also be conjugated directly to signalgenerating compounds, e.g., by conjugation with an enzyme orfluorophore. Enzymes of interest as labels will primarily be hydrolases,particularly phosphatases, esterases and glycosidases, oroxidoreductases, particularly peroxidases. Fluorescent compounds includefluorescein and its derivatives, rhodamine and its derivatives, dansyl,umbelliferone, etc. Chemiluminescent compounds include luciferin, and0.2,3-dihydrophthalazinediones, e.g., luminol. For a review of variouslabeling or signal producing systems which may be used, see, U.S. Pat.No. 4,391,904, which is incorporated herein by reference.

[0120] Some assay formats do not require the use of labeled components.For instance, agglutination assays can be used to detect the presence ofthe target antibodies. In this case, antigen-coated particles areagglutinated by samples comprising the target antibodies. In thisformat, none of the components need be labeled and the presence of thetarget antibody is detected by simple visual inspection.

[0121] The proteins of the present invention can be used for identifyingcompounds that bind to (e.g., substrates), and/or increase or decrease(i.e., modulate) the enzymatic activity of catalytically activepolypeptides of the present invention. The method comprises contacting apolypeptide of the present invention with a compound whose ability tobind to or modulate enzyme activity is to be determined. The polypeptideemployed will have at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or95% of the specific activity of the native, full-length polypeptide ofthe present invention (e.g., enzyme). Methods of measuring enzymekinetics are well known in the art. See, e.g., Segel, BiochemicalCalculations, 2^(nd) ed., John Wiley and Sons, New York (1976).

[0122] Antibodies can be raised to a protein of the present invention,including individual, allelic, strain, or species variants, andfragments thereof, both in their naturally occurring (full-length) formsand in recombinant forms. Additionally, antibodies are raised to theseproteins in either their native configurations or in non-nativeconfigurations. Anti-idiotypic antibodies can also be generated. Manymethods of making antibodies are known to persons of skill.

[0123] In some instances, it is desirable to prepare monoclonalantibodies from various mammalian hosts, such as mice, rodents,primates, humans, etc. Description of techniques for preparing suchmonoclonal antibodies are found in, e.g., Basic and Clinical Immunology,4th ed., Stites et al., Eds., Lange Medical Publications, Los Altos,Calif., and references cited therein; Harlow and Lane, Supra; Goding,Monoclonal Antibodies: Principles and Practice, 2^(nd) ed., AcademicPress, New York, N.Y. (1986); and Kohler and Milstein, Nature256:495-497 (1975).

[0124] Other suitable techniques involve selection of libraries ofrecombinant antibodies in phage or similar vectors (see, e.g., Huse etal., Science 246:1275-1281 (1989); and Ward et al., Nature 341:544-546(1989); and Vaughan et al., Nature Biotechnology 14:309-314 (1996)).Alternatively, high avidity human monoclonal antibodies can be obtainedfrom transgenic mice comprising fragments of the unrearranged humanheavy and light chain Ig loci (i.e., minilocus transgenic mice).Fishwild et al., Nature Biotech. 14:845-851 (1996). Also, recombinantimmunoglobulins may be produced. See, Cabilly, U.S. Pat. No. 4,816,567;and Queen et al., Proc. Natl. Acad. Sci. U.S.A. 86:10029-10033 (1989).

[0125] The antibodies of this invention can be used for affinitychromatography in isolating proteins of the present invention, forscreening expression libraries for particular expression products suchas normal or abnormal protein or for raising anti-idiotypic antibodieswhich are useful for detecting or diagnosing various pathologicalconditions related to the presence of the respective antigens.

[0126] Frequently, the proteins and antibodies of the present inventionmay be labeled by joining, either covalently or non-covalently, asubstance which provides for a detectable signal. A wide variety oflabels and conjugation techniques are known and are reported extensivelyin both the scientific and patent literature. Suitable labels includeradionucleotides, enzymes, substrates, cofactors, inhibitors,fluorescent moieties, chemiluminescent moieties, magnetic particles, andthe like.

Transformation of Cells

[0127] The method of transformation is not critical to the presentinvention; various methods of transformation are currently available. Asnewer methods are available to transform crops or other host cells theymay be directly applied. Accordingly, a wide variety of methods havebeen developed to insert a DNA sequence into the genome of a host cellto obtain the transcription and/or translation of the sequence to effectphenotypic changes in the organism. Thus, any method which provides forefficient transformation/transfection may be employed.

[0128] A DNA sequence coding for the desired polynucleotide of thepresent invention, for example a cDNA or a genomic sequence encoding afull length protein, can be used to construct an expression cassettewhich can be introduced into the desired plant. Isolated nucleic acidacids of the present invention can be introduced into plants accordingto techniques known in the art. Generally, expression cassettes asdescribed above and suitable for transformation of plant cells areprepared.

[0129] Techniques for transforming a wide variety of higher plantspecies are well known and described in the technical, scientific, andpatent literature. See, for example, Weising et al., Ann. Rev. Genet.22:421-477 (1988). For example, the DNA construct may be introduceddirectly into the genomic DNA of the plant cell using techniques such aselectroporation, PEG poration, particle bombardment, silicon fiberdelivery, or microinjection of plant cell protoplasts or embryogeniccallus. See, e.g., Tomes et al., Direct DNA Transfer into Intact PlantCells Via Microprojectile Bombardment. pp.197-213 in Plant Cell, Tissueand Organ Culture, Fundamental Methods, Eds. O. L. Gamborg and G. C.Phillips, Springer-Verlag Berlin Heidelberg New York, 1995.Alternatively, the DNA constructs may be combined with suitable T-DNAflanking regions and introduced into a conventional Agrobacteriumtumefaciens host vector. The virulence functions of the Agrobacteriumtumefaciens host will direct the insertion of the construct and adjacentmarker into the plant cell DNA when the cell is infected by thebacteria. See, U.S. Pat. No. 5,591,616.

[0130] The introduction of DNA constructs using polyethylene glycolprecipitation is described in Paszkowski et al., Embo J. 3:2717-2722(1984). Electroporation techniques are described in Fromm et al., Proc.Natl. Acad. Sci. U.S.A. 82:5824 (1985). Ballistic transformationtechniques are described in Klein et al., Nature 327:70-73 (1987).

[0131]Agrobacterium tumefaciens-meditated transformation techniques arewell described in the scientific literature. See, for example Horsch etal., Science 233:496-498 (1984), and Fraley et al., Proc. Natl. Acad.Sci. 80:4803 (1983). For instance, Agrobacterium transformation of maizeis described in U.S. Patent No. 5,981,840. Agrobacterium transformationof soybean is described in U.S. Pat. No. 5,563,055.

[0132] Other methods of transformation include (1) Agrobacteriumrhizogenes-mediated transformation (see, e.g., Lichtenstein and FullerIn: Genetic Engineering, Vol. 6, P. W. J. Rigby, Ed., London, AcademicPress, 1987; and Lichtenstein, C. P. and Draper, J. In: DNA Cloning,Vol. 11, D. M. Glover, Ed., Oxford, IRI Press, 1985), ApplicationPCT/US87/02512 (WO 88/02405 published Apr. 7,1988) describes the use ofA. rhizogenes strain A4 and its Ri plasmid along with A. tumefaciensvectors pARC8 or pARC16, (2) liposome-mediated DNA uptake (see, e.g.,Freeman et al., Plant Cell Physiol. 25:1353 (1984)), and (3) thevortexing method (see, e.g., Kindle, Proc. Natl. Acad. Sci. USA 87:1228(1990)).

[0133] DNA can also be introduced into plants by direct DNA transferinto pollen as described by Zhou et al., Methods in Enzymology 101:433(1983); D. Hess, Intern Rev. Cytol., 107:367 (1987); Luo et al., PlantMol. Biol. Reporter 6:165 (1988). Expression of polypeptide codingpolynucleotides can be obtained by injection of the DNA intoreproductive organs of a plant as described by Pena et al., Nature325:274 (1987). DNA can also be injected directly into the cells ofimmature embryos and the rehydration of desiccated embryos as describedby Neuhaus et al., Theor. Appl. Genet. 75:30 (1987); and Benbrook etal., in Proceedings Bio Expo 1986, Butterworth, Stoneham, Mass., pp.27-54 (1986).

[0134] Animal and lower eukaryotic (e.g., yeast) host cells arecompetent or rendered competent for transformation by various means.There are several well-known methods of introducing DNA into animalcells. These include: calcium phosphate precipitation, fusion of therecipient cells with bacterial protoplasts containing the DNA, treatmentof the recipient cells with liposomes containing the DNA, DEAE dextran,electroporation, biolistics, and micro-injection of the DNA directlyinto the cells. The transfected cells are cultured by means well knownin the art. Kuchler, R. J., Biochemical Methods in Cell Culture andVirology, Dowden, Hutchinson and Ross, Inc. (1977).

Transgenic Plant Regeneration

[0135] Transformed plant cells which are derived by any of the abovetransformation techniques can be cultured to regenerate a whole plantwhich possesses the transformed genotype. Such regeneration techniquesoften rely on manipulation of certain phytohormones in a tissue culturegrowth medium, typically relying on a biocide and/or herbicide markerthat has been introduced together with a polynucleotide of the presentinvention. For transformation and regeneration of maize see, Gordon-Kammet al., The Plant Cell 2:603-618 (1990).

[0136] Plants cells transformed with a plant expression vector can beregenerated, e.g., from single cells, callus tissue or leaf discsaccording to standard plant tissue culture techniques. It is well knownin the art that various cells, tissues, and organs from almost any plantcan be successfully cultured to regenerate an entire plant. Plantregeneration from cultured protoplasts is described in Evans et al.,Protoplasts Isolation and Culture, Handbook of Plant Cell Culture,Macmillan Publishing Company, New York, pp.124-176 (1983); and Binding,Regeneration of Plants, Plant Protoplasts, CRC Press, Boca Raton, pp.21-73 (1985).

[0137] The regeneration of plants containing the foreign gene introducedby Agrobacterium can be achieved as described by Horsch et al., Science,227:1229-1231 (1985) and Fraley et al., Proc. Natl. Acad. Sci. U.S.A.80:4803 (1983). This procedure typically produces shoots within two tofour weeks and these transformant shoots are then transferred to anappropriate root-inducing medium containing the selective agent and anantibiotic to prevent bacterial growth. Transgenic plants of the presentinvention may be fertile or sterile.

[0138] Regeneration can also be obtained from plant callus, explants,organs, or parts thereof. Such regeneration techniques are describedgenerally in Klee et al., Ann. Rev. Plant Phys. 38:467-486 (1987). Theregeneration of plants from either single plant protoplasts or variousexplants is well known in the art. See, for example, Methods for PlantMolecular Biology, A. Weissbach and H. Weissbach, eds., Academic Press,Inc., San Diego, Calif. (1988). For maize cell culture and regenerationsee generally, The Maize Handbook, Freeling and Walbot, Eds., Springer,New York (1994); Corn and Corn Improvement, 3^(rd) edition, Sprague andDudley Eds., American Society of Agronomy, Madison, Wis. (1988).

[0139] One of skill will recognize that after the expression cassette isstably incorporated in transgenic plants and confirmed to be operable,it can be introduced into other plants by sexual crossing. Any of anumber of standard breeding techniques can be used, depending upon thespecies to be crossed.

[0140] In vegetatively propagated crops, mature transgenic plants can bepropagated by the taking of cuttings, via production of apomictic seed,or by tissue culture techniques to produce multiple identical plants.Selection of desirable transgenics is made and new varieties areobtained and propagated vegetatively for commercial use. In seedpropagated crops, mature transgenic plants can be self crossed toproduce a homozygous inbred plant. The inbred plant produces seedcontaining the newly introduced heterologous nucleic acid. These seedscan be grown to produce plants that would produce the selectedphenotype.

[0141] Parts obtained from the regenerated plant, such as flowers,seeds, leaves, branches, fruit, and the like are included in theinvention, provided that these parts comprise cells comprising theisolated nucleic acid of the present invention. Progeny and variants,and mutants of the regenerated plants are also included within the scopeof the invention, provided that these parts comprise the introducednucleic acid sequences.

[0142] Transgenic plants expressing a selectable marker can be screenedfor transmission of the nucleic acid of the present invention by, forexample, standard immunoblot and DNA detection techniques. Transgeniclines are also typically evaluated on levels of expression of theheterologous nucleic acid. Expression at the RNA level can be determinedinitially to identify and quantitate expression-positive plants.Standard techniques for RNA analysis can be employed and include PCRamplification assays using oligonucleotide primers designed to amplifyonly the heterologous RNA templates and solution hybridization assaysusing heterologous nucleic acid-specific probes. The RNA-positive plantscan then be analyzed for protein expression by Western immunoblotanalysis using the specifically reactive antibodies of the presentinvention. In addition, in situ hybridization and immunocytochemistryaccording to standard protocols can be done using heterologous nucleicacid specific polynucleotide probes and antibodies, respectively, tolocalize sites of expression within transgenic tissue. Generally, anumber of transgenic lines are usually screened for the incorporatednucleic acid to identify and select plants with the most appropriateexpression profiles.

[0143] Transgenic plants of the present invention can be homozygous forthe added heterologous nucleic acid; i.e., a transgenic plant thatcontains two added nucleic acid sequences, one gene at the same locus oneach chromosome of a chromosome pair. A homozygous transgenic plant canbe obtained by sexually mating (selfing) a heterozygous transgenic plantthat contains a single added heterologous nucleic acid, germinating someof the seed produced and analyzing the resulting plants produced foraltered expression of a polynucleotide of the present invention relativeto a control plant (i.e., native, non-transgenic). Back-crossing to aparental plant and out-crossing with a non-transgenic plant are alsocontemplated. Alternatively, propagation of heterozygous transgenicplants could be accomplished through apomixis.

[0144] The present invention provides a method of genotyping a plantcomprising a polynucleotide of the present invention. Genotypingprovides a means of distinguishing homologs of a chromosome pair and canbe used to differentiate segregants in a plant population. Molecularmarker methods can be used for phylogenetic studies, characterizinggenetic relationships among crop varieties, identifying crosses orsomatic hybrids, localizing chromosomal segments affecting monogenictraits, map based cloning, and the study of quantitative inheritance.See, e.g., Plant Molecular Biology: A Laboratory Manual, Chapter 7,Clark, Ed., Springer-Verlag, Berlin (1997). For molecular markermethods, see generally, The DNA Revolution by Andrew H. Paterson 1996(Chapter 2) in: Genome Mapping in Plants (ed. Andrew H. Paterson) byAcademic Press/R. G. Landis Company, Austin, Tex., pp.7-21.

[0145] The particular method of genotyping in the present invention mayemploy any number of molecular marker analytic techniques such as, butnot limited to, restriction fragment length polymorphisms (RFLPs). RFLPsare the product of allelic differences between DNA restriction fragmentscaused by nucleotide sequence variability. Thus, the present inventionfurther provides a means to follow segregation of a gene or nucleic acidof the present invention as well as chromosomal sequences geneticallylinked to these genes or nucleic acids using such techniques as RFLPanalysis.

[0146] Plants which can be used in the method of the invention includemonocotyledonous and dicotyledonous plants. Preferred plants includemaize, wheat, rice, barley, oats, sorghum, millet, rye, soybean,sunflower, safflower, alfalfa, canola, cotton, or turf grass.

[0147] Seeds derived from plants regenerated from transformed plantcells, plant parts or plant tissues, or progeny derived from theregenerated transformed plants, may be used directly as feed or food, orfurther processing may occur.

[0148] All publications cited in this application are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

[0149] The present invention will be further described by reference tothe following detailed examples. It is understood, however, that thereare many extensions, variations, and modifications on the basic theme ofthe present invention beyond that shown in the examples and description,which are within the spirit and scope of the present invention.

[0150] Other objects, features, advantages and aspects of the presentinvention will become apparent to those of skill from the followingdescription. It should be understood, however, that the followingdescription and the specific examples, while indicating certainembodiments of the invention, are given by way of illustration only.Various changes and modifications within the spirit and scope of thedisclosed invention will become readily apparent to those skilled in theart from reading the following description and from reading the otherparts of the present disclosure.

EXAMPLES Example 1 cDNA Library Construction

[0151] A. Total RNA Isolation

[0152] Total RNA was isolated from maize tissues with TRizol Reagent(Life Technology Inc. Gaithersburg, Md.) using a modification of theguanidine isothiocyanate/acid-phenol procedure described by Chomczynskiand Sacchi (Anal. Biochem. 162:156 (1987)). In brief, plant tissuesamples were pulverized in liquid nitrogen before the addition of theTRIzol Reagent, and then were further homogenized with a mortar andpestle. Addition of chloroform followed by centrifugation was conductedfor separation of an aqueous phase and an organic phase. The total RNAwas recovered by precipitation with isopropyl alcohol from the aqueousphase. Good results have been obtained using tissues such asnight-harvested earshoot with husk at stage V-12 from corn line B73,corn night-harvested leaf tissue at stage V8-V10 from line B73, cornanther tissue at prophase I from line B73, 4 DAP coenocytic embryo sacsfrom corn line B73, 67 day old corn cob from corn line L, and corn BMSsuspension cells treated with chemicals related to phosphatases.

[0153] B. Poly(A)+RNA Isolation

[0154] The selection of poly(A)+RNA from total RNA was performed usingPolyATract system (Promega Corporation. Madison, Wis.). In brief,biotinylated oligo(dT) primers were used to hybridize to the 3′ poly(A)tails on mRNA. The hybrids were captured using streptavidin coupled toparamagnetic particles and a magnetic separation stand. The mRNA waswashed at high stringent condition and eluted by RNase-free deionizedwater.

[0155] C. cDNA Library Construction

[0156] cDNA synthesis was performed and unidirectional cDNA librarieswere constructed using the SuperScript Plasmid System (Life TechnologyInc. Gaithersburg, Md.). The first stand of cDNA was synthesized bypriming an oligo(dT) primer containing a NotI site. The reaction wascatalyzed by SuperScript Reverse Transcriptase II at 45° C. The secondstrand of cDNA was labeled with alpha-³²P-dCTP and a portion of thereaction was analyzed by agarose gel electrophoresis to determine cDNAsizes. cDNA molecules smaller than 500 base pairs and unligated adapterswere removed by Sephacryl-S400 chromatography. The selected cDNAmolecules were ligated into pSPORT1 vector in between NotI and SalIsites.

Example 2 Sequencing and cDNA Subtraction Procedures Used for MaizeEST's

[0157] A. Sequencing Template Preparation

[0158] Individual colonies were picked and DNA was prepared either byPCR with M13 forward primers and M13 reverse primers, or by plasmidisolation. All the cDNA clones were sequenced using M13 reverse primers.

[0159] B. Q-bot Subtraction Procedure

[0160] cDNA libraries subjected to the subtraction procedure were platedout on 22×22 cm² agar plate at density of about 3,000 colonies perplate. The plates were incubated in a 37° C. incubator for 12-24 hours.Colonies were picked into 384-well plates by a robot colony picker,Q-bot (GENETIX Limited). These plates were incubated overnight at 37° C.

[0161] Once sufficient colonies were picked, they were pinned onto 22×22cm2 nylon membranes using Q-bot. Each membrane contained 9,216 coloniesor 36,864 colonies. These membranes were placed onto individual agarplates with appropriate antibiotic. The plates were incubated at 37° C.for overnight.

[0162] After colonies were recovered on the second day, these filterswere placed on filter paper prewefted with denaturing solution for fourminutes, then were incubated on top of a boiling water bath foradditional four minutes. The filters were then placed on filter paperprewefted with neutralizing solution for four minutes. After excesssolution was removed by placing the filters on dry filter papers for oneminute, the colony side of the filters were place into Proteinase Ksolution, incubated at 37° C. for 40-50 minutes. The filters were placedon dry filter papers to dry overnight. DNA was then cross-linked tonylon membrane by UV light treatment.

[0163] Colony hybridization was conducted as described by Sambrook, J.,Fritsch, E. F. and Maniatis, T., (in Molecular Cloning: A LaboratoryManual, 2^(nd) Edition). The following probes were used in colonyhybridization:

[0164] 1. First strand cDNA from the same tissue from which the librarywas made to remove the most redundant clones.

[0165] 2. 48-192 most redundant cDNA clones from the same library basedon previous sequencing data.

[0166] 3. 192 most redundant cDNA clones in the entire corn sequencedatabase.

[0167] 4. A Sal-A20 oligonucleotide: TCG ACC CAC GCG TCC GAA AAA AAA AAAAAA AAA AAA, removes clones containing a poly A tail but no cDNA. SeeSEQ ID NO: 26.

[0168] 5. cDNA clones derived from rRNA.

[0169] The image of the autoradiography was scanned into computer andthe signal intensity and cold colony addresses of each colony wasanalyzed. Re-arraying of cold-colonies from 384 well plates to 96 wellplates was conducted using Q-bot.

Example 3 Identification and Isolation of ITPK Genes Using PCR

[0170] The maize ITPK-3, -5, and -6 genomic clones exemplified by SEQ IDNOS: 3, 7 and 9 respectively, were isolated by PCR using thecommercially available Roche Expand High Fidelity PCR System. TemplateDNA was isolated using the CTAB method of Example 5C. The forward primerof SEQ ID NO: 15 was used with reverse primers SEQ ID NOS: 16, 17, 22,or 27 to amplify the ITPK-5 gene from various maize lines. The bufferand polymerase concentrations were used as defined for the kit with theDNA concentrations and cycling conditions as follows:

[0171] DNA Concentrations:

[0172] 500 ng template DNA and 0.3 μM primers in a 50 μl PCR reactionmixture containing

[0173] 200 μM dNTPs in buffer and polymerase provided by the Roche kit.Thermocycling conditions are as follows (#cycles):

[0174] 1 cycle: denature 2 min. at 94° C.

[0175] 10 cycles: denature 15 sec. at 94° C.

[0176]  anneal 30 sec. at 55° C.

[0177]  elongate 60 sec. at 68° C.

[0178] 15 cycles: denature 15 sec. at 94° C.

[0179]  anneal 30 sec at 55° C.

[0180]  elongate 60 sec.+5 sec. each cycle at 68° C.

[0181] 1 cycle: elongate 7 min. at 72° C.

[0182] The products of the PCR reaction were analyzed on agarose gelsusing standard molecular biology techniques.

[0183] Similar to the Arabidopsis genomic clone (TIGR Arabidopsisdatabase, At5g16760), it was found that the maize B73 ITPK-5 genomicsequence has no introns.

Example 4 Vector Construction

[0184] All vectors are constructed using standard molecular biologytechniques used by those of skill in the art (Sambrook et al., supra).

[0185] A. Vectors for Plant Transformation

[0186] Vectors were constructed for plant transformation using eitherparticle bombardment or Agrobacterium transformation protocols.

[0187] Plasmids were constructed by inserting ITPK-5 expressioncassettes, including the following: oleosin promoter::ITPK-5::nosterminator, oleosin promoter::Shl intron::ITPK-5::nos terminator,oleosin promoter::ubiquitin intron::ITPK-5::nos terminator or globulin5promoter::ITPK-5::globulin1 terminator, into a descendent plasmid ofpSB11 which contains the BAR expression cassette. Both the ITPK-5 andthe BAR expression cassettes were located between the right and leftborders of the T-DNA.

[0188] For example, the Zea mays ITPK-5 coding region, including the 5′UTR and 3′ UTR was isolated from a full length PCR clone as a 1.4 kbXhoI/SacI fragment. The fragment was inserted in-frame into aSacI/SalI-digested plasmid between the globulins promoter andterminator. The globulins promoter::ITPK-5::globulin1 terminatortranscription unit was moved as a 2.94 kb HindIII/HpaI fragment into asecond intermediate vector in order to flank the transcription unit withBstEII sites. These BstEII sites were used to excise the fragment andinsert it into a binary vector containing the BAR selectable marker. TheITPK-5 cassette is linked to the selectable marker between the right andleft borders of the T-DNA. This vector was used for insert preparationfor particle gun transformation as well as for generating Agrobacteriumtransformation vectors as described below. In this case, insert DNA forparticle gun transformation was generated by isolating the 6.6 kb PmeIfragment from the vector.

[0189] In other examples, ITPK-5 cassettes were linked withtranscription units for the Zea mays inositol polyphosphate kinase(IPPK) or myo-inositol 1-phosphate synthase (MIIIPS-3) polynucleotidessimilarly constructed for expression in the maize embryo. IPPKpolynucleotide sequences are disclosed in U.S. Application Serial No.10/042,894 filed Jan. 9, 2002, M1 PS polynucleotide sequences aredisclosed in WO 99/05298, the contents of which are herein incorporatedby reference in their entirety. Alternatively, convenient restrictionsites were used to fuse portions of the ITPK-5 coding sequence withportions of the coding sequence of IPPK or other ITPK polynucleotides togenerate chimeric transcripts. Such stacked or linked expressioncassettes were also inserted into derivatives of pSB11 with the BARselectable marker as described above.

[0190] The plasmid pSB11 was obtained from Japan Tobacco Inc. (Tokyo,Japan). The construction of pSB11 from pSB21 and the construction ofpSB21 from starting vectors is described by Komari et al. (1996, PlantJ. 10:165-174). The T-DNA of the plasmid was integrated in to thesuperbinary plasmid pSB1 (Saito et al. EP 672 752 A.1) by homologousrecombination between the two plasmids. The plasmid pSB1 was alsoobtained from Japan Tobacco Inc. These plasmids were either used forparticle bombardment transformation, or for Agrobacterium-mediatedtransformation after making a cointegrate in an appropriateAgrobacterium strain as described more fully below.

[0191] Competent cells of the Agrobacterium strain LBA4404 harboringpSB1 were created using the protocol as described by Lin (1995) inMethods in Molecular Biology, ed. Nickoloff, J. A. (Humana Press,Totowa, N.J.). The plasmid containing the expression cassettes waselectroporated into competent cells of the Agrobacterium strain LBA4404harboring pSB1 to create the cointegrate plasmid in Agrobacterium. Cellsand DNA were prepared for electroporation by mixing 1 μl of plasmid DNA(˜100 ng) with 20 μl of competent Agrobacterium cells in a 0.2 cmelectrode gap cuvette (Bio-Rad Cat# 165-2086, Hercules, Calif.).Electroporation was performed in a Bio-Rad Micropulser (Cat# 165-2100,Hercules, Calif.) using the EC2 setting, which delivers 2.5 kV to thecells. Successful recombination was verified by restriction analysis ofthe plasmid after transformation of the cointegrate plasmid back into E.coli DH5α cells.

[0192] B. Vectors for In vitro Protein Expression in E. coli

[0193] Vectors were constructed for protein expression of ITPK-2, ITPK-3and ITPK-5 (SEQ ID NOS: 1, 3, and 7) in E. coli using standardprotocols. Each ITPK sequence was fused with GST to produce GST-taggedproteins.

[0194] Cloning sites were introduced into the ITPK-2 sequence by PCRwith the primers of SEQ ID NOS: 18 and 19. The primer of SEQ ID NO: 18introduces a SmaI site to the 5′ end of the ITPK-2 sequence, while theprimer of SEQ ID NO: 19 introduces a NotI site to the 3′ end of thesequence. Using these restriction sites, the ITPK-2 sequence was clonedinto the pGEX-4T-2 vector (PHARMACIA BIOTECH) to generate the ITPK-2GST-tagged expression vector PHP16334.

[0195] In the same way, the ITPK-3 GST-tagged expression constructPHP16335 was made using PCR primer SEQ ID NOS: 20 and 21 to introduce a5′ SmaI site and a 3′ NotI site to the ITPK-3 sequence.

[0196] The ITPK-5 GST-tagged expression construct was made by firstusing the primer pair of SEQ ID NOS: 15 and 22 to generate the ITPK-5insert. This insert was cloned into the pCR vector (INVITROGEN TACloning kit). The ITPK-5 pCR vector was digested with EcoRI and clonedinto the pGEX-4T-1 vector (Pharmacia Biotech). Insert orientation wasconfirmed using a restriction enzyme digest.

[0197] These expression vectors were used to transform E. coli strainDH5α using standard techniques. The expression of GST-tagged ITPKproteins and assay for substrate-specificity is further described inExample 7.

Example 5 Plant Transformation

[0198] A. Particle Bombardment Transformation and Regeneration of MaizeCallus

[0199] Immature maize embryos from greenhouse or field grown High type11 donor plants are bombarded with a plasmid comprising an ITPKpolynucleotide of the invention operably linked to an appropriatepromoter. If the polynucleotide does not include a selectable marker,another plasmid containing a selectable marker gene can beco-precipitated on the particles used for bombardment. For example, aplasmid containing the PAT gene (Wohlleben et al. (1988) Gene 70:25-37)which confers resistance to the herbicide Bialaphos can be used.Transformation is performed as follows.

[0200] The ears are surface sterilized in 50% Chlorox bleach plus 0.5%Micro detergent for 20 minutes, and rinsed two times with sterile water.The immature embryos are excised and placed embryo axis side down(scutellum side up), 25 embryos per plate. These are cultured on 560Lagar medium 4 days prior to bombardment in the dark. Medium 560L is anN6-based medium containing Eriksson's vitamins, thiamine, sucrose,2,4-D, and silver nitrate. The day of bombardment, the embryos aretransferred to 560Y medium for 4 hours and are arranged within the2.5-cm target zone. Medium 560Y is a high osmoticum medium (560L withhigh sucrose concentration).

[0201] A plasmid vector comprising a polynucleotide of the inventionoperably linked to the selected promoter is constructed. This plasmidDNA, plus plasmid DNA containing a PAT selectable marker if needed, isprecipitated onto 1.1 μm (average diameter) tungsten pellets using aCaCl₂ precipitation procedure as follows: 100 μl prepared tungstenparticles (0.6 mg) in water, 20 μl (2 μg) DNA in TrisEDTA buffer (1 μgtotal), 100 μl 2.5 M CaCl₂, 40 μl 0.1 M spermidine.

[0202] Each reagent is added sequentially to the tungsten particlesuspension. The final mixture is sonicated briefly. After theprecipitation period, the tubes are centrifuged briefly, liquid removed,washed with 500 ml 100% ethanol, and centrifuged again for 30 seconds.Again the liquid is removed, and 60 μl 100% ethanol is added to thefinal tungsten particle pellet. For particle gun bombardment, thetungsten/DNA particles are briefly sonicated and 5 μl spotted onto thecenter of each macrocarrier and allowed to dry about 2 minutes beforebombardment.

[0203] The sample plates are bombarded at a distance of 8 cm from thestopping screen to the tissue, using a DuPont biolistics helium particlegun. All samples receive a single shot at 650 PSI, with a total of tenaliquots taken from each tube of prepared particles/DNA.

[0204] Four to 12 hours post bombardment, the embryos are moved to 560P(a low osmoticum callus initiation medium similar to 560L but with lowersilver nitrate), for 3-7 days, then transferred to 560R selectionmedium, an N6 based medium similar to 560P containing 3 mg/literBialaphos, and subcultured every 2 weeks. After approximately 10 weeksof selection, callus clones are sampled for PCR and activity of thepolynucleotide of interest. Positive lines are transferred to 288Jmedium, an MS-based medium with lower sucrose and hormone levels, toinitiate plant regeneration. Following somatic embryo maturation (2-4weeks), well-developed somatic embryos are transferred to medium forgermination and transferred to the lighted culture room. Approximately7-10 days later, developing plantlets are transferred to medium in tubesfor 7-10 days until plantlets are well established. Plants are thentransferred to inserts in flats (equivalent to 2.5″ pot) containingpotting soil and grown for 1 week in a growth chamber, subsequentlygrown an additional 1-2 weeks in the greenhouse, then transferred toClassic™ 600 pots (1.6 gallon) and grown to maturity. Plants aremonitored for expression of the polynucleotide of interest.

[0205] B. Agrobacterium-Mediated Transformation and Regeneration ofMaize Callus

[0206] For Agrobacterium-mediated transformation of maize, an ITPKpolynucleotide sequence of the present invention is used with the methodof Zhao (U.S. Patent No. 5,981,840, and PCT patent publicationWO98/32326; the contents of which are hereby incorporated by reference).

[0207] Briefly, immature embryos are isolated from maize and the embryoscontacted with a suspension of Agrobacterium containing a polynucleotideof the present invention, where the bacteria are capable of transferringthe nucleotide sequence of interest to at least one cell of at least oneof the immature embryos (step 1: the infection step). In this step theimmature embryos are immersed in an Agrobacterium suspension for theinitiation of inoculation. The embryos are co-cultured for a time withthe Agrobacterium (step 2: the co-cultivation step). The immatureembryos are cultured on solid medium following the infection step.Following this co-cultivation period an optional “resting” step iscontemplated. In this resting step, the embryos are incubated in thepresence of at least one antibiotic known to inhibit the growth ofAgrobacterium without the addition of a selective agent for planttransformants (step 3: resting step). The immature embryos are culturedon solid medium with antibiotic, but without a selecting agent, forelimination of Agrobacterium and for a resting phase for the infectedcells. Next, inoculated embryos are cultured on medium containing aselective agent and growing transformed callus is recovered (step 4: theselection step). The immature embryos are cultured on solid medium witha selective agent resulting in the selective growth of transformedcells. The callus is then regenerated into plants (step 5: theregeneration step), and calli grown on selective medium are cultured onsolid medium to regenerate the plants.

[0208] C. Transformation of Dicots with Transgene

[0209] An expression cassette, with an ITPK polynucleotide of thepresent invention operably linked to appropriate regulatory elements forexpression, can be introduced into embryogenic suspension cultures ofsoybean by particle bombardment using essentially the methods describedin Parroft, W. A., L. M. Hoffman, D. F. Hildebrand, E. G. Williams, andG. B. Collins, (1989) Recovery of primary transformants of soybean,Plant Cell Rep. 7:615-617. This method, with modifications, is describedbelow.

[0210] Seed is removed from pods when the cotyledons are between 3 and 5mm in length. The seeds are sterilized in a bleach solution (0.5%) for15 minutes after which time the seeds are rinsed with sterile distilledwater. The immature cotyledons are excised by first cutting away theportion of the seed that contains the embryo axis. The cotyledons arethen removed from the seed coat by gently pushing the distal end of theseed with the blunt end of the scalpel blade. The cotyledons are thenplaced (flat side up) SB1 initiation medium (MS salts, B5 vitamins, 20mg/L 2,4-D, 31.5 g/l sucrose, 8 g/L TC Agar, pH 5.8). The Petri platesare incubated in the light (16 hr day; 75-80 μE) at 26° C. After 4 weeksof incubation the cotyledons are transferred to fresh SB1 medium. Afteran additional two weeks, globular stage somatic embryos that exhibitproliferative areas are excised and transferred to FN Lite liquid medium(Samoylov, V. M., D. M. Tucker, and W. A. Parrott (1998) Soybean[Glycine max (L.) Merrill] embryogenic cultures: the role of sucrose andtotal nitrogen content on proliferation. In Vitro Cell Dev. Biol.-Plant34:8-13). About 10 to 12 small clusters of somatic embryos are placed in250 ml flasks containing 35 ml of SB172 medium. The soybean embryogenicsuspension cultures are maintained in 35 mL liquid media on a rotaryshaker, 150 rpm, at 26° C. with florescent lights (20 μE) on a 16:8 hourday/night schedule. Cultures are sub-cultured every two weeks byinoculating approximately 35 mg of tissue into 35 mL of liquid medium.

[0211] Soybean embryogenic suspension cultures are then transformedusing particle gun bombardment (Klein et al. (1987) Nature (London)327:70; U.S. Pat. No. 4,945,050). A BioRad Biolistic™ PDS1000/HEinstrument can be used for these transformations. A selectable markergene, which is used to facilitate soybean transformation, is a chimericgene composed of the 35S promoter from Cauliflower Mosaic Virus (Odellet al. (1985) Nature 313:810-812), the hygromycin phosphotransferasegene from plasmid pJR225 (from E. coli; Gritz et al. (1983) Gene25:179-188) and the 3′ region of the nopaline synthase gene from theT-DNA of the Ti plasmid of Agrobacterium tumefaciens.

[0212] To 50 μL of a 60 mg/mL 1 μm gold particle suspension is added (inorder): 5 μL DNA (1 μg/μL), 20 μl spermidine (0.1 M), and 50 μL CaCl₂(2.5 M). The particle preparation is agitated for three minutes, spun ina microfuge for 10 seconds and the supernatant removed. The DNA-coatedparticles are washed once in 400 μL 70% ethanol and resuspended in 40 μLof anhydrous ethanol. The DNA/particle suspension is sonicated threetimes for one second each. Five μL of the DNA-coated gold particles arethen loaded on each macro carrier disk.

[0213] Approximately 300-400 mg of a two-week-old suspension culture isplaced in an empty 60×15 mm petri dish and the residual liquid removedfrom the tissue with a pipette. Membrane rupture pressure is set at 1100psi and the chamber is evacuated to a vacuum of 28 inches mercury. Thetissue is placed approximately 8 cm away from the retaining screen, andis bombarded three times. Following bombardment, the tissue is dividedin half and placed back into 35 ml of FN Lite medium.

[0214] Five to seven days after bombardment, the liquid medium isexchanged with fresh medium. Eleven days post bombardment the medium isexchanged with fresh medium containing 50 mg/mL hygromycin. Thisselective medium is refreshed weekly. Seven to eight weeks postbombardment, green, transformed tissue will be observed growing fromuntransformed, necrotic embryogenic clusters. Isolated green tissue isremoved and inoculated into individual flasks to generate new, clonallypropagated, transformed embryogenic suspension cultures. Each new lineis treated as an independent transformation event. These suspensions arethen subcultured and maintained as clusters of immature embryos, ortissue is regenerated into whole plants by maturation and germination ofindividual embryos.

[0215] D. DNA Isolation from Callus and Leaf Tissues

[0216] In order to screen putative transformation events for thepresence of the transgene, genomic DNA is extracted from calli or leavesusing a modification of the CTAB (cetyltriethylammonium bromide, SigmaH5882) method described by Stacey and Isaac (1994). Approximately100-200 mg of frozen tissues is ground into powder in liquid nitrogenand homogenised in 1 ml of CTAB extraction buffer (2% CTAB, 0.02 M EDTA,0.1 M Tris-Cl pH 8, 1.4 M NaCl, 25 mM DTT) for 30 min at 65° C.Homogenised samples are allowed to cool at room temperature for 15 minbefore a single protein extraction with approximately 1 ml 24:1 v/vchloroform:octanol is done. Samples are centrifuged for 7 min at 13,000rpm and the upper layer of supernatant collected using wide-mouthedpipette tips. DNA is precipitated from the supernatant by incubation in95% ethanol on ice for 1 h. DNA threads are spooled onto a glass hook,washed in 75% ethanol containing 0.2 M sodium acetate for 10 min,air-dried for 5 min and resuspended in TE buffer. Five μl RNAse A isadded to the samples and incubated at 37° C. for 1 h.

[0217] For quantification of genomic DNA, gel electrophoresis isperformed using a 0.8% agarose gel in 1×TBE buffer. One microlitre ofthe samples are fractionated alongside 200, 400, 600 and 800 ng μ⁻¹ λuncut DNA markers.

Example 6 Identification of High Phosphorus/Low Phytate Transgenic CornLines

[0218] The resulting transformants are screened for inorganic phosphorusand/or phytate levels using the calorimetric assays as described below.The extraction procedure used is compatible with both assays. Thecolorimetric assays can be performed sequentially or simultaneously.Putative events are usually initially screened for increased levels ofinorganic phosphorous compared to wild type control and then furthercharacterized by the phytate assay.

[0219] A. Sample Preparation

[0220] Individual kernels are crushed to a fine powder using a ball millgrinding device. Grinding of certain samples, for example high oil cornlines, can be facilitated by chilling the sample in the grindingapparatus at −80° C. for 2 hours prior to grinding. Transfer 25-35 mg ofeach ground sample to new 1.5 ml microfuge tube. Extract each samplewith 1 ml of 0.4N hydrochloric acid (HCl) for 3.5 hours at roomtemperature with shaking to keep the meal suspended. Transfer 1 ml ofthis suspension to a 1.1 ml Megatiter tube (Cat# 2610, Continental Labs)and place into the 96-well Megatiter plate (Cat# 2405, ContinentalLabs). Clarify the extract by low-speed centrifugation, for example 4000rpm for 15 minutes in a Jouan centrifuge. The clarified supernatant isused for the assays described in sections 6B and 6C. below.

[0221] B. Quantitative Inorganic Phosphate Assay

[0222] This assay is performed in duplicate for each sample according tothe method of Chen, et al. (1956 Anal. Chem. 28:1756-1758) with somemodifications. For each sample, mix a 200 μl aliquot of clarifiedextract with 100 μl 30% trichloroacetic acid (TCA). Clarify by low speedcentrifugation at 3900×g for 10 min. Transfer 50 μl clarifiedsupernatant to a new 96-well microtiter plate. Add 100 μl of the colorreagent (7 parts 0.42% ammonium molybdate in 1 N H₂SO₄: 1 part 10%ascorbic acid) and incubate at 37° C. for 30 minutes. A phosphatestandard curve is generated using NaH₂PO₄ in the range of 0-160 nmoldiluted from a 10 mM stock solution in 2 parts 0.4N HCl: 1 part 30% TCA.Measure the absorbance at 800 nm.

[0223] C. Quantitative Phytate Assay

[0224] This assay is modified from Haug and Lantzsch (1983) J. Sci. FoodAgric. 34:1423-1426. This assay is performed in duplicate for eachsample. Phytate standard (Cat# P-7660, Sigma Chemical Co., St. Louis,Mo.) stock solution is made by dissolving 150 mg phytate in 100 mldistilled water (DDW). Standards in the range of 0-35 μg/ml are made bydilution with 0.2N HCl. Samples are prepared in 96-well microtiterplates by mixing 35 μl of clarified supernatant (from 6A) with 35 μl ofDDW, add 140 μl ferric solution (0.2 g ammonium iron (III) sulphatedodecahydrate (Merck Art 3776)/liter in 0.2N HCl). Plates are sealed andincubated for 30 minutes at 99° C., then cooled to 4° C. Plates are keptin an ice-water bath for 15 minutes then transferred to room temperaturefor 20 minutes. Centrifuge the plates at low speed to pelletprecipitate, for example spin 30 minutes at 4000 rpm in a Jouancentrifuge. After centrifugation transfer 80 μl clarified supernatant toa new 96-well plate and mix with 120 μl 1% 2,2′-bipyridine-1%thioglycollic acid solution (10 g 2,2′-bipyridine (Merck Art. 3098), 10ml thioglycolic acid (Merck Art. 700) in DDW to 1 liter). The absorbanceat 519 nm is read using a VERSAmax microplate reader (Molecular Devices,Sunnyvale, Calif.).

[0225] Each plant identified as a potential high available phosphorustransgenic is tested again to confirm the original elevated phosphorusreading. Confirmed high available phosphorous lines are selected on thebasis of uniformity for the trait. Transformants which are positive withthe calorimetric assays can then be subjected to more rigorous analysesto include Southern, Northern and Western blotting and/or quantitationand identification of phytic acid and inositol phosphate intermediatesby HPLC.

Example 7 Determining the Substrate Specificity of the ITPK clones

[0226] A. Expression of ITPK and Purification

[0227] A single colony of E. coli strain DH5α containing a GST-taggedITPK expression vector described in Example 4 is cultured overnight at37° C. in LB medium containing ampicillin (Amp). The overnight cultureis diluted 1:10 with fresh LB+Amp and incubated at 37° C. with vigorousagitation until the A600 reading of the culture is in the range of 0.6-2O.D. units. GST fusion protein expression is induced by the addition ofIPTG to the culture to a final concentration of 50 μM. The cultures areincubated at 37° C. with agitation for an additional 3 hrs.

[0228] Cells are harvested by centrifugation at 7,700×g for 10 minutesat 4° C. Cell pellets are resuspended in ice-cold bacterial lysis buffer(50 mM Tris-HCl, pH 7,4, 100 mM NaCl, 100 μM phenylmethylsulfonylfluoride). The cells are lysed on ice by sonication, then Triton X-100is added to a final concentration of 1%. After incubation on ice for 1hour, the lysate is clarified by centrifugation at 12,000×g for 10minutes at 4° C. The GST-ITPK proteins are affinity purified by batchabsorption to Glutathione Sepharose 4B bead resin (Bulk GST Purificationkit, Pharmacia Biotech) at a ratio of 1 ml bed volume of the 50%Glutathione Sepharose 4B slurry per 100 ml clarified lysate. The mixtureis incubated 45 minutes at 4° C. with gentle shaking. Following theconditions detailed in the manufacturer's instructions, the beads arewashed four times in lysis buffer, then two times in phosphate bufferedsaline. GST-tagged ITPK protein is eluted with 10 mM reduced glutathionein 50 mM Tris-HCl (pH 8.0), 100 mM NaCl. For every 500 ml of cellculture, 200 μl buffer is used to elute the protein. After elution,glycerol is added to a final concentration of 50% and purified GST-ITPKproteins are stored in 50% glycerol at −20° C.

[0229] B. Assay for ITPK Activity and Substrate Specificity

[0230] Inositol phosphate kinase activities are assayed according toWilson and Majerus (1996 J. Biol. Chem. 271:11904-11910) with somemodifications. This assay cannot identify the stereospecific structureof the inositol phosphate product, but it does demonstrate the inositolphosphate kinase activity of the protein of interest.

[0231] Purified GST-ITPK fusion proteins are used in an inositol1,3,4-trisphosphate 5/6-kinase activity assay. The activity assay isperformed in a volume of 25 μl. The assay mixture contains 20 mM HEPES,pH 7.2, 6 mM MgCl₂, 10 mM LiCl, 1 mM DTT, 40 μM Ins(1,3,4)P₃, 40 μM ATP,0.5 μl γ-³²P-ATP (3000 Ci/mmol) and 5 μl enzyme per reaction. Thereaction mixture is incubated at 30° C., or room temperature, for 30minutes. The reaction is stopped by the addition of 2.8 μl stoppingsolution (3M HCl, 2M KH₂PO₄) to the 25 μl reaction. One microlitersamples of each reaction, along with Ins(1,3,4,5)P₄ and Ins(1,3,4,6)P₄standards, are separated on a polyethyleneimine (PEI)-cellulose thinlayer chromatography plate (Merck) with 0.5M HCl according to Spencer etal. (In Methods in Inositide Research, (1990) pp. 39-43, Ed. R. F.Irvine, Raven Press, NY). After separation, the TLC plate is air-driedat 70° C., wrapped in plastic wrap and exposed to X-ray film to detectthe ³²P-labelled reaction products. The reaction products are quantifiedby cutting the spot out of the TLC plate and measuring the radioactivityin a liquid scintillation counter. The identity of the reaction productis confirmed by comparing the distance migrated to the migration of theInsP₄ standard controls run on each TLC plate. In addition to theIns(1,3,4)P₃, other inositol phosphate substrates are also tested todetermine the substrate specificity of the ITPK enzymes. The othersubstrates tested under the same conditions above included: Ins(1)P,Ins(2)P, Ins(4)P, Ins(1,4)P₂, Ins(4,5)P₂, Ins(3,5,6)P₃, Ins(1,4,5)P₃,Ins(2,4,5)P₃, Ins(3,4,5,6)P₄, Ins(1,3,4,6)P₄, Ins(1,3,5,6)P₄,Ins(1,2,5,6)P₄, Ins(1,3,4,5)P₄, and Ins(1,3,4,5,6)P₅.

[0232] Assay results indicated that each of ITPK-2, ITPK-3 and ITPK-5are capable of phosphorylating the Ins(1,3,4)P₃ substrate to produce³²P-labelled products that comigrate with Ins(1,3,4,5)P₄ andIns(1,3,4,6)P₄ on PEI-cellulose TLC plates, confirming the expectedactivity of the enzymes. Further, the ITPKs tested could also useIns(3,5,6)P₃, Ins(3,4,5,6)P₄ and Ins(1,2,5,6)P₄ as a substrate in the invitro assay. When Ins(3,4,5,6)P₄ was used as a substrate, the productcomigrated with Ins(1,3,4,5,6)P₅, indicating the enzyme can also act asa Ins(3,4,5,6)P₄ 1-kinase. The Ins(3,4,5,6) 1-kinase activity was alsoreported for a human ITPK enzyme (Yang, X. and Shears, S. B. (2000)Biochem J. 351:551-555; Ho et al. (2002) Curr Biol 12:477-482).Ins(1,4,5)P₃-kinase activity has been reported for Ins(1,3,4)P₃5/6-kinase in Entamoeba histolytica (Field et al. (2000) Mol BiochemParasitol 108:119-123). When the substrate Ins(1,3,4,5)P₄ was used withITPK-5, weak kinase activity was detected and 2 products, an InsP₄ andan InsP₅, were produced. No kinase activity was detected when any ofIns(1)P, Ins(2)P, Ins(4)P, Ins(1,4)P₂, Ins(4,5)P₂, Ins(1,4,5)P₃,Ins(2,4,5)P₃, Ins(1,3,4,6)P₄, Ins(1,3,5,6)P₄, or Ins(1,3,4,5,6)P₅ wereused as substrates in the reaction mixture.

Example 8 ITPK Corn Knockout Mutants

[0233] Mu-tagged corn populations (TUSC) (Bensen, RJ, et al. (1995)Plant Cell 7:75-84) are screened for knockouts of the ITPK-5 gene (SEQID NO: 7), using the primers of SEQ ID NO: 23 or 24 paired with aMu-primer SEQ ID NO: 25 in PCR reactions. From a collection of about40,000 Mu-insertion lines, four independent lines were identified ashaving a Mu-insertion in the ITPK-5 gene, these lines are designatedITPK5-Mum1, ITPK5-Mum2, ITPK5-Mum3, and ITPK5-Mum4, or ITPK5-Mumcollectively. Kernels from three of these lines were screened forphytate and inorganic phosphate levels versus phytate mutants Lpal andLpa2, as well as wild type controls, using the assays described inExample 6.

[0234] Analysis of individual ITPK5-Mum F2 seeds for phytic acid andorganic phosphate (Pi) showed that about 25% of F2 seeds had a reducedlevel of phytic acid and an increased level of P_(i), while 75% of F2seeds showed phytic acid and Pi at wildtype levels. Similar phenotypesin four independent lines and this segregation ratio support theassumption that the low phytic acid phenotype is caused by Mu-insertionin the maize ITPK-5 gene.

[0235] The Mu-insertion was mapped for ITPK5-Mum lines by sequencing theMu-ITPK-5 junction region. In ITPK5-Mum1, Mu is inserted at nucleotideposition 237, which is amino acid position 61. The Mu insertion inITPK5-Mum2 occurs at nucleotide position 245, which is amino acidposition 64. In ITPK5-Mum3, the Mu insertion occurs at nucleotideposition 366, which corresponds to amino acid 104. The Mu insertion inITPK5-Mum4 is at nucleotide position 872, which is amino acid 273. Inall lines, mapping demonstrated that Mu insertion disrupted the ITPK-5open reading frame.

[0236] Genotyping individual F2 seeds confirmed that Mu was inserted inthe ITPK-5 gene. Individual F2 seeds were ground to a fine powder. Analiquot of each meal was used to determine phytic acid and P_(i), andthe remaining meal used for DNA extraction and PCR analysis. PCR wasdone using the primer pair of SEQ ID NO: 15 and SEQ ID NO: 27, whichflank the Mu insertion site. A PCR product of 1.3 KB is expected to beamplified from the intact ITPK-5 gene, but not from the ITPK5-Mumallele. Seeds with an ITPK-5/ITPK-5 or ITPK-5/ITPK5-Mum genotype willyield the 1.3 kb PCR fragment, but ITPK5-Mum/ITPK5-Mum seeds will not.It was found that the low phytic acid kernels did not contain an intactcopy of the ITPK-5 gene, while the 1.3 kb DNA fragment was onlyamplified from kernels with normal phytic acid and P_(i) levels. F3 andsubsequent generations also showed the low phytic acid and high P_(i)phenotype in all four ITPK5-Mum lines.

[0237] HPLC analyses for myo-inositol (Example 9) and inositolphosphates (Example 10) showed that in addition to the changes in kernelphytic acid and P_(i), ITPK5-Mum lines also accumulate myo-inositol,InsP₃, InsP₄, and InsP₅ in the embryo. No obvious differences were foundin the endosperm.

[0238] Results indicate that relative to the wild type control, phytatewas reduced by about 30%. Further, it was observed that inorganicphosphorous was increased to about 0.6 mg/g in the ITPK-5 TUSC line vs.0.16 mg/g for the wild type control. Myo-inositol levels were increasedabove 170 μg/g in ITPK5-Mum vs. about 75-90 μg/g in the normal wholekernel control. In embryos only, myo-inositol levels were increased toabout 438 μg/g in ITPK5-Mum vs. 254 μg/g in the wildtype control.

[0239] ITPK5-Mum lines have a phenotype very similar to Ipa2 mutants(Raboy et al. (2000) Plant Physiol. 124:355-368). Lpa2 is a recessive,low phytic acid mutant created by chemical mutagenesis, this mutant alsoaccumulates InsP₃, InsP₄, InsP₅ and P_(i) in the seeds. We also foundthat the embryo of Ipa2 mutant seeds accumulate myo-inositol, similar tothe ITPK5-Mum lines, to about 614 μg/g. No gene or genes have beenidentified as being responsible for the Ipa2 phenotype. Crosses betweenITPK5-Mum3 and Ipa2-1 and Ipa2-2 were performed to determine if thegenes are allelic.

[0240] Before crossing, all lines were backcrossed with inbreds toreduce background effects. ITPK5-Mum3 was crossed with maize inbred linePHP38 to reduce Mu copy number. The ITPK5-Mum3 allele was tracked bymonitoring the low phytate phenotype of corresponding selfed ears. Afterthree backcrosses, the ITPK5-Mum3 line was selfed to produce ITPK5-Mum3homozygotes. Lpa2 mutant lines were provided by Victor Raboy. The Ipa2-1mutant allele line (Raboy et al. (2000) Plant Physiol. 124:355-368) wasbackcrossed twice with maize inbred PHJ90, then selfed to homozygosity.A second, separately isolated Ipa2 mutant allele line, Ipa2-2 (Raboy,personal communication), was backcrossed four times to inbred linePHN46, then selfed to homozygosity.

[0241] When homozygous ITPK5-Mum3 plants were crossed with the recessiveIpa2-1 and Ipa2-2 mutant lines, all the Fl seeds displayed the lowphytic acid and high P_(i) phenotype. When heterozygous ITPK5-Mum3plants were crossed with lpa2-2, the F1 seeds showed 1:1 mutant:wildtypephenotype segregation. This demonstrates that the Ipa2 mutant is amutation in the ITPK-5 gene.

[0242] The ITPK-5 gene was amplified from the lpa2-2 mutant using theprimer pair of SEQ ID NO: 15 and SEQ ID NO: 27. Sequencing of theamplified DNA showed a point mutation of C to T at nucleotide position158 (SEQ ID NO: 28). This mutation introduces a stop codon (TAG) atamino acid 35 instead of the glutamine (Gln) found in normal ITPK-5.Thus, Ipa2-2 appears to produce a severely truncated 34 amino acidpolypeptide (SEQ ID NO: 29) which lacks inositol phosphate kinaseactivity based on the phenotype of the seeds.

[0243] PCR amplification of the ITPK-5 gene from the Ipa2-1 allele lineswith the same primer pair did not produce any product. Southern analysisrevealed differing band patterns between Ipa2-1 mutant vs. non-mutantnear-isogenic lines using a 0.7 kb probe which covered nucleotides367-1088. This region contains a BamHI restriction site and, asexpected, two bands are detected in the non-mutant lines (˜3.7 kb and-1.4 kb). While the Ipa2-1 mutant line also showed two bands, thefragment were significantly larger (both >˜8 kb). Restriction enzymesEcoRI, EcoRV, HindIII, and XbaI are absent from the probe region and, asexpected, a single band was detected in the non-mutant line. However,XbaI digestion of Ipa2-1 mutant line reveals two fragments. HindIIIdigestion also produced two fragments (˜0.7 kb and ˜1.6 kb) not seen inthe non-mutant ITPK-5 gene. These results indicate a rearrangement ofthe genomic sequence in the ITPK-5 locus of the Ipa2-1 mutant, likelyproducing the loss of an intact ITPK-5 gene in the mutant. RT-PCR wasdone on Ipa2-1 immature seeds, but no transcript could be detected.

Example 9 Myo-inositol assay

[0244] Putative events can also be screened to determine the effect thetransgene may have on myo-inositol levels in the kernel using a gaschromatography/mass spectrometry method. Either whole, mature, drykernels or excised embryos are assayed. Embryos are dissected frommature whole kernels after soaking dry seeds in double distilled water(DDW) four hours at 4° C. The isolated embryos are lyophilized, thenground for extraction as described below.

[0245] Whole, mature, dry kernels or embryos are ground to a fine mealin a ball mill apparatus. Each sample is analyzed in triplicate. Forextraction, three aliquots of each sample is extracted with 50% v/vethyl alcohol (1:1100% ethyl alcohol:DDW) at a ratio of 0.1 g meal/1 ml50% ethyl alcohol at room temperature for one hour with vigorousshaking. The extract supernatant is decanted and filtered through a 0.45μm syringe filter. The meal residue is re-extracted with fresh 50%ethanol following the same procedure, combining the two filtrates. Eachsample is vortexed, and a 1 ml aliquot taken and evaporated to drynessin a SpeedVac at medium heat.

[0246] A myo-inositol standard stock of 10 mg/ml is made in doubledistilled water (DDW) which is used to make a 1 mg/ml standard solutionworking stock. Aliquots of 50 μl, 100 μl, 200 μl and 300 μl aretransferred to new tubes and evaporated to dryness in a SpeedVac asdescribed above. This calibration set covers a concentration range of 5μg/ml to 30 μg/ml of each component.

[0247] Thoroughly dried standards and samples are resuspended in 50 μlpyridine. To this, 50 μl of 100:1trimethylsilylimadazole-trimethylchlorosilane (TMSI-TMCS) is added toeach sample. Samples are compromised if a precipitate forms. Tubes aresealed, vortexed and incubated 15 min. at 60° C. After incubation, 1 mlof 2,2,4-trimethylpentane and 0.5 ml DDW are added. Vortex samples andcentrifuge at low speed (2000 rpm) 1000×g for 5 minutes. The top,organic layer is transferred to a 2 ml autosampler vial and crimpsealed. At this point, the samples can be stored at 4° C. untilanalyzed.

[0248] Samples are analyzed on a Hewlett-Packard 5890/7673/5972 GasChromatography/Mass Spectrometry (GC/MS) apparatus using aHewlett-Packard 30 m×0.25 mm i.d.×0.25 μm film thickness 5MS columnunder the following conditions:

[0249] Inlet temperature: 250° C.

[0250] Injection Volume: 1 ml

[0251] Split Ratio: Splitless

[0252] Oven Temperature: 70° C. initial, hold for 2 min.

[0253]  Ramp at 25° C./min. to 170° C., hold for 0 min.

[0254]  Ramp at 5° C./min. to 215° C., hold for 0 min.

[0255]  Ramp at 25° C./min to 250° C., hold for 5 min.

[0256]  23.4 min. total run time

[0257] Detector Temperature: 250° C.

[0258] Carrier Gas: Helium, 36.6 cm/sec at 70° (1 ml/min.)

[0259] Full scan (m/z 50-650), acquired at −70 eV after 5 minute solventdelay. Results are reported as μg/ml for the final sample analyzed bythe GC/MS, this concentration is multiplied by a factor of 20 beforeusing to calculate μg/g dry weight tissue. The moisture content of themature kernels is determined from a separate aliquot of eachexperimental sample so that the results can be adjusted to a dry weightbasis.

[0260] Myo-inositol levels are quantified as follows:$\frac{{µg}\quad \text{myo-inositol}}{\text{g~~dry~~wt.~~tissue}} = {\frac{{µg}\quad ({X20})}{\text{ml~~sample}} \times \frac{1\quad {ml}\quad \text{sample}}{1\quad {ml}\quad \text{extract}} \times \frac{10\quad {ml}\quad \text{extract}}{0.5\quad g\quad \text{tissue}}}$

[0261] Regression coefficients of four-point calibration curves weretypically 0.999-1.000.

Example 10 HPLC of Phytate and Inositol Phosphate Intermediates

[0262] Phytate and inositol phosphate intermediates associated withphytic acid in wheat, corn, and soybean seeds can be identified andquantitated using gradient anion-exchange chromatography HPLC withconductivity detection. While phytate and intermediate inositolphosphates can be identified using this method, the method practicedcurrently has been optimized for phytate, it is not optimized forquantitation of intermediate inositol phosphates. For other HPLCseparations of inositol phosphates see also Anonymous, (1990) “Analysisof inositol phosphates” Dionex Corp. Application Note AN 65; Xu, P.,Price, J., and Aggett, P. (1992) Progress in Food and Nutrition Science16:245262; Rounds, M. A. and Nielsen, S. S. (1993) J.Chromatogr653:148-152; and Trugo, L. and von Baer, D. (1998) Associationfor animal production, publication 93:1128. Inositol phosphates can alsobe identified by thin-layer chromatographic methods, see for exampleSpencer, C. E. L et al. (1990) Ch. 4 in Methods in Inositide Research,Ed. R. F. Irving, Raven Press, Ltd., NY pp. 39-43; and Hatzack, F. andRasmussen, S. K. (1999) J. Chromatogr B 736:221-229.

[0263] For anion-exchange HPLC, a phytic acid standard range of 0.25,0.5, 1.0, 2.0 and 3.0 mg/ml is prepared in 0.4M hydrochloric acid (HCl)from a 20 mg/ml working stock in 0.4M HCl. Seed samples are prepared bygrinding seeds to a fine meal in a ball mill grinding apparatus.Replicate aliquots are weighed and extracted in 0.4M HCl in a ratio of0.1 g meal/1 ml 0.4M HCl. Usually 5 ml 0.4M HCl is used to extract 0.5 gcorn or wheat meal while 15 ml 0.4M HCl is used to extract 1.5 g soymeal. After the addition of the extraction buffer, the samples areextracted with moderate-vigorous shaking for 2 hrs. at room temperature,then transferred to 4° C. overnight without shaking. The supernatantsfrom corn and wheat are clarified by low-speed centrifugation at 1000×gfor 10 minutes. Due to the high fat content, the low-speed supernatantfrom soy sample extracts is further clarified by ultracentrifugation at55,000 rpm at 4° C. for 1 hour. After ultracentrifugation, the clear,middle layer is removed with a needle or extended tip disposabletransfer pipette. Clarified samples are filtered through a 0.45 μmsyringe filter and stored at 40° C. until analysis. Just beforeanalysis, an aliquot of each sample is filtered with a MilliporeDurapore ULTRAFREE-MC 0.22 μm centrifugal filter unit, or equivalent.

[0264] Using a Dionex DX500 HPLC with a Dionex model AS3500 autosampler,25 microliter samples are subjected to anion-exchange HPLC separation bya linear gradient of 0.06-0.118M sodium hydroxide (NaOH) in 1% isopropylalcohol on a Dionex 4×250 mm OmniPac PAX-100 column at a flow rate of 1ml/min. Dionex 4×50 mm OmniPac PAX-100 guard and ATC-1 anion trapcolumns were used. The total run time is 30 min. with data collectionfrom 0 to 20 minutes. A Dionex conductivity detector module was usedwith a Dionex ASRS-Ultra II anion self-regenerating suppressor in theexternal water mode. Signal collection is set at 0.5 Hz, detector unitsin μS, current at 300 mA, with the Detection Stablilizer regulated at30° C. and temperature compensation at 1.7.

[0265] Soybean samples appear to cause column performance deterioration,therefore it is helpful to interject short column cleaning run betweensamples. The cleaning run comprises a series of injections for 1M HCl,1M NaOH, and 90% acetonitrile.

[0266] Chromatographic traces show that InsP₃, InsP₄, and InsP₅ arepartially, but clearly resolved from each other and InsP₆.

Example 11 ITPK5 mRNA Expression

[0267] Northern blotting analysis is conducted to reveal anydevelopmental and/or tissue specific mRNA expression patterns for ITPK5.The analysis is conducted using standard molecular biology protocolssuch as those found in Current Protocols in Molecular Biology (Ausubelet al., Eds., Greene Publishing and Wiley-Interscience, New York, 1995);Plant Molecular Biology: A Laboratory Manual (Clark, Ed.,Springer-Verlag, Berlin, 1997); and Molecular Cloning: A LaboratoryManual, 2^(nd) Ed. (Sambrook et al., Cold Spring Harbor Laboratory, Vols1-3, 1989), herein incorporated by reference.

[0268] Northern blotting analysis was conducted using total RNA atdifferent developmental stages from various tissues from corn line B73.The tissues tested include 7 and 11 DAP whole kernel, 15, 22, 29, and 35DAP endosperm, 15, 22, 29, and 35 DAP embryo, stalk, stem, leaf, silk, 0DAP cob, brace root, and husk tissues. RNA was prepared using thePurescript RNA isolation kit (Gentra, Minneapolis, Minn.). Tenmicrograms of RNA were resolved on 1% agarose/formaldehyde/MOPS gels andtransferred to nylon membranes using standard conditions. Preparation ofthe ITPK5 probe, hybridization, and washing were carried out accordingto the manufacturer's instructions.

[0269] The maize ITPK5 gene transcript expression peaks in the embryo at15 days after pollination (DAP). Expression could be detected in embryosat earlier stages, but at very low levels. Expression peaks at 15 DAP,then declines at later stages. No expression was detected from endospermor vegetative tissues.

[0270] The above examples are provided to illustrate the invention butnot to limit its scope. Other variants of the invention will be readilyapparent to one of ordinary skill in the art and are encompassed by theappended claims. All publications, patents, patent applications, andcomputer programs cited herein are hereby incorporated by reference.

1 29 1 1458 DNA Zea mays CDS (224)...(1270) 1 gcacgagcct tgccctgcccgcacacacca cctgtccccg cacggccgca ccgctccgct 60 cccgaggctt agggctccggccgcgctccc ttcccttccc ggcattcccg atctctcgcc 120 gccgcccgcc tggccttgatctcgatcgcc ctcccctctc cgctctcgct ccggcaggcc 180 ggcccgggtt tgtctcgccgctattgggcc tcggcacgct gcg atg gtg tcg ggc 235 Met Val Ser Gly 1 gtg tgcgtg ggg acg gag ggg cag gtg gac cct gag gcg gtg gcg ccg 283 Val Cys ValGly Thr Glu Gly Gln Val Asp Pro Glu Ala Val Ala Pro 5 10 15 20 gct gtcgcg gag gag gcg gtg gtg ccg gcg ccc gcg agg gag gtc gtg 331 Ala Val AlaGlu Glu Ala Val Val Pro Ala Pro Ala Arg Glu Val Val 25 30 35 gtg ggg tacgcg ctc acg agt aag aag gcc aag agc ttc ctc caa ccc 379 Val Gly Tyr AlaLeu Thr Ser Lys Lys Ala Lys Ser Phe Leu Gln Pro 40 45 50 aag ctc cgg gggctc gcc agg aaa aag gga atc ttg ttt gtc gct att 427 Lys Leu Arg Gly LeuAla Arg Lys Lys Gly Ile Leu Phe Val Ala Ile 55 60 65 gat cag aaa cgc ccattg tct gat caa ggt cca ttt gac att gtt ctt 475 Asp Gln Lys Arg Pro LeuSer Asp Gln Gly Pro Phe Asp Ile Val Leu 70 75 80 cat aag ttg act gga aggggg tgg cag caa ttg ctg gag gaa tat agg 523 His Lys Leu Thr Gly Arg GlyTrp Gln Gln Leu Leu Glu Glu Tyr Arg 85 90 95 100 gag gca cac cca gaa gttact gtt ctt gat cca cct ggc gca ata gca 571 Glu Ala His Pro Glu Val ThrVal Leu Asp Pro Pro Gly Ala Ile Ala 105 110 115 aac ttg ctt gat cgc cagtct atg ctt caa gaa gta tct gaa ttg gac 619 Asn Leu Leu Asp Arg Gln SerMet Leu Gln Glu Val Ser Glu Leu Asp 120 125 130 ctc aca gat tgt cat ggtaaa gta cgt gtg cct aag cag cta ttc gtt 667 Leu Thr Asp Cys His Gly LysVal Arg Val Pro Lys Gln Leu Phe Val 135 140 145 aat act gat ccc tca tcaata cca gct gca gtt agg agg gca ggt ctc 715 Asn Thr Asp Pro Ser Ser IlePro Ala Ala Val Arg Arg Ala Gly Leu 150 155 160 tct ctc cca ttg gtg gcaaaa ccc ttg gtg gcg aag tcc cat gag cta 763 Ser Leu Pro Leu Val Ala LysPro Leu Val Ala Lys Ser His Glu Leu 165 170 175 180 tcc ctt gct tat gatcca act tca ctg acc aaa ctt gag ccc ccc ttg 811 Ser Leu Ala Tyr Asp ProThr Ser Leu Thr Lys Leu Glu Pro Pro Leu 185 190 195 gtt ctt cag gaa tttgtt aac cat ggt ggc gtc atg ttt aag gtg tac 859 Val Leu Gln Glu Phe ValAsn His Gly Gly Val Met Phe Lys Val Tyr 200 205 210 att gtt ggg gat gcaata agg gtt gta cgt cga ttt tca ctc cca aat 907 Ile Val Gly Asp Ala IleArg Val Val Arg Arg Phe Ser Leu Pro Asn 215 220 225 gtt gat gaa ggc gatcta tcg aac aat gct ggg gta ttt cgg ttt cca 955 Val Asp Glu Gly Asp LeuSer Asn Asn Ala Gly Val Phe Arg Phe Pro 230 235 240 agg gtc tct tgt gctgca gcc agt gca gat gat gca gat ctt gac cct 1003 Arg Val Ser Cys Ala AlaAla Ser Ala Asp Asp Ala Asp Leu Asp Pro 245 250 255 260 cgt gtt gct gaactt cct ccg aga cca ttg ctt gag atc ttg gca cga 1051 Arg Val Ala Glu LeuPro Pro Arg Pro Leu Leu Glu Ile Leu Ala Arg 265 270 275 gag ctg cgc cgacga ctg ggt ctt aga cta ttc aac atc gat atg att 1099 Glu Leu Arg Arg ArgLeu Gly Leu Arg Leu Phe Asn Ile Asp Met Ile 280 285 290 agg gag cat gggacg cga gat cgg ttt tat gtc ata gac atg aac tac 1147 Arg Glu His Gly ThrArg Asp Arg Phe Tyr Val Ile Asp Met Asn Tyr 295 300 305 ttt cct ggg tacggc aaa atg cct gga tac gag cac gtg ttc acc gac 1195 Phe Pro Gly Tyr GlyLys Met Pro Gly Tyr Glu His Val Phe Thr Asp 310 315 320 ttc ctg ctg agcctt gcc aag aaa gag tac aag aga cga caa agc tat 1243 Phe Leu Leu Ser LeuAla Lys Lys Glu Tyr Lys Arg Arg Gln Ser Tyr 325 330 335 340 agc tcg ctaagc tca ggc gaa tgg tga taagcgagga gactactcgg 1290 Ser Ser Leu Ser SerGly Glu Trp * 345 cggggcatgt atatgtctat catccacgat gcgtgcgtac agatgtacttgtgcatgacg 1350 agagataatg ggtcgtagaa gcggagggct gttgtcaggc tataactaactgttgcttta 1410 catgtgctaa ctgttgatgc ttcagaataa attttgtttg ggtggaaa1458 2 348 PRT Zea mays 2 Met Val Ser Gly Val Cys Val Gly Thr Glu GlyGln Val Asp Pro Glu 1 5 10 15 Ala Val Ala Pro Ala Val Ala Glu Glu AlaVal Val Pro Ala Pro Ala 20 25 30 Arg Glu Val Val Val Gly Tyr Ala Leu ThrSer Lys Lys Ala Lys Ser 35 40 45 Phe Leu Gln Pro Lys Leu Arg Gly Leu AlaArg Lys Lys Gly Ile Leu 50 55 60 Phe Val Ala Ile Asp Gln Lys Arg Pro LeuSer Asp Gln Gly Pro Phe 65 70 75 80 Asp Ile Val Leu His Lys Leu Thr GlyArg Gly Trp Gln Gln Leu Leu 85 90 95 Glu Glu Tyr Arg Glu Ala His Pro GluVal Thr Val Leu Asp Pro Pro 100 105 110 Gly Ala Ile Ala Asn Leu Leu AspArg Gln Ser Met Leu Gln Glu Val 115 120 125 Ser Glu Leu Asp Leu Thr AspCys His Gly Lys Val Arg Val Pro Lys 130 135 140 Gln Leu Phe Val Asn ThrAsp Pro Ser Ser Ile Pro Ala Ala Val Arg 145 150 155 160 Arg Ala Gly LeuSer Leu Pro Leu Val Ala Lys Pro Leu Val Ala Lys 165 170 175 Ser His GluLeu Ser Leu Ala Tyr Asp Pro Thr Ser Leu Thr Lys Leu 180 185 190 Glu ProPro Leu Val Leu Gln Glu Phe Val Asn His Gly Gly Val Met 195 200 205 PheLys Val Tyr Ile Val Gly Asp Ala Ile Arg Val Val Arg Arg Phe 210 215 220Ser Leu Pro Asn Val Asp Glu Gly Asp Leu Ser Asn Asn Ala Gly Val 225 230235 240 Phe Arg Phe Pro Arg Val Ser Cys Ala Ala Ala Ser Ala Asp Asp Ala245 250 255 Asp Leu Asp Pro Arg Val Ala Glu Leu Pro Pro Arg Pro Leu LeuGlu 260 265 270 Ile Leu Ala Arg Glu Leu Arg Arg Arg Leu Gly Leu Arg LeuPhe Asn 275 280 285 Ile Asp Met Ile Arg Glu His Gly Thr Arg Asp Arg PheTyr Val Ile 290 295 300 Asp Met Asn Tyr Phe Pro Gly Tyr Gly Lys Met ProGly Tyr Glu His 305 310 315 320 Val Phe Thr Asp Phe Leu Leu Ser Leu AlaLys Lys Glu Tyr Lys Arg 325 330 335 Arg Gln Ser Tyr Ser Ser Leu Ser SerGly Glu Trp 340 345 3 1655 DNA Zea mays CDS (246)...(1286) 3 gaattcccgggtcgacccac gcgtccggcc attattaaca gctccgcggt ccctccctcc 60 ctcggtcggtcggcgtcggt ccctccctcc ccacccagtt agtcctcagc ctatcccgtg 120 cccgcgcagagcaccgcctc tgctcgaccc accaccctct gtgcagagtt aattaacctt 180 gaggtttccgattgcccttc ccttccgctc ctctcgccca ttcgcggcga gattcagcgg 240 caagg atg cgcctg cac gcg gag gtg cgg gat gag atg gag gag ggg agc 290 Met Arg Leu HisAla Glu Val Arg Asp Glu Met Glu Glu Gly Ser 1 5 10 15 gag gag ggg gctgtg acg gct tcg gcg ggg ctg tcg cca ccg cca ctc 338 Glu Glu Gly Ala ValThr Ala Ser Ala Gly Leu Ser Pro Pro Pro Leu 20 25 30 atc ggt gcg gcg gcgccg gtt ccc cgg cta gtg gtg ggg ttc gcc ctc 386 Ile Gly Ala Ala Ala ProVal Pro Arg Leu Val Val Gly Phe Ala Leu 35 40 45 acg aag aag aag gtg aagagc ttc ctg cag ccc aag ctg ctc ctg ctg 434 Thr Lys Lys Lys Val Lys SerPhe Leu Gln Pro Lys Leu Leu Leu Leu 50 55 60 gcc agg aag aat gga atc agtttt gta tct att gat gag tct ctt ccc 482 Ala Arg Lys Asn Gly Ile Ser PheVal Ser Ile Asp Glu Ser Leu Pro 65 70 75 ctc tca gaa caa ggc cct ttt gatgtt att tta cac aag att act agg 530 Leu Ser Glu Gln Gly Pro Phe Asp ValIle Leu His Lys Ile Thr Arg 80 85 90 95 aag gag tgg cag aag gtt ctg gaggac tat cac gaa gaa cat cca gaa 578 Lys Glu Trp Gln Lys Val Leu Glu AspTyr His Glu Glu His Pro Glu 100 105 110 gtt act gtc ctt gac cca cca aatgct atc gag cat ctg aac aat cga 626 Val Thr Val Leu Asp Pro Pro Asn AlaIle Glu His Leu Asn Asn Arg 115 120 125 caa tca atg ctt gaa gaa gta gctgat ttg aac ctg tca aat ttc tat 674 Gln Ser Met Leu Glu Glu Val Ala AspLeu Asn Leu Ser Asn Phe Tyr 130 135 140 gga gaa gtt tgt atc cca cgc cagctg gtc att acg aaa gat cca tcc 722 Gly Glu Val Cys Ile Pro Arg Gln LeuVal Ile Thr Lys Asp Pro Ser 145 150 155 tct ata cca act tct gta gct atggct gga cta act ttg ccc ttg gtt 770 Ser Ile Pro Thr Ser Val Ala Met AlaGly Leu Thr Leu Pro Leu Val 160 165 170 175 gcc aag cca ttg gtt gtt gatggg acg tct aaa ggt cat gaa cta tat 818 Ala Lys Pro Leu Val Val Asp GlyThr Ser Lys Gly His Glu Leu Tyr 180 185 190 ctt gca tat gac gag gca tccttg tca atg ctt gat ccg cct ctg gtt 866 Leu Ala Tyr Asp Glu Ala Ser LeuSer Met Leu Asp Pro Pro Leu Val 195 200 205 cta cag gaa ttc ata aac catggc ggg atc ctc ttt aag gtg tat atc 914 Leu Gln Glu Phe Ile Asn His GlyGly Ile Leu Phe Lys Val Tyr Ile 210 215 220 att ggt gaa aca ata cag gttgtc cgc agg ttc tct ctt cct gat gtt 962 Ile Gly Glu Thr Ile Gln Val ValArg Arg Phe Ser Leu Pro Asp Val 225 230 235 aac aca tat gac tta cta aacaac gtt ggc atc tat cga ttg cca aga 1010 Asn Thr Tyr Asp Leu Leu Asn AsnVal Gly Ile Tyr Arg Leu Pro Arg 240 245 250 255 gtt tca tgt gct gca gctagt gcg gat gat gca gat ctt gac cct ctt 1058 Val Ser Cys Ala Ala Ala SerAla Asp Asp Ala Asp Leu Asp Pro Leu 260 265 270 att gca gag ctt cct ccaagg cca ctt cta gag aaa ctg ggc agg gag 1106 Ile Ala Glu Leu Pro Pro ArgPro Leu Leu Glu Lys Leu Gly Arg Glu 275 280 285 ctt cgt ggc cgg ctt ggtttg aga ttg ttc aat ata gat atg att aga 1154 Leu Arg Gly Arg Leu Gly LeuArg Leu Phe Asn Ile Asp Met Ile Arg 290 295 300 gaa ctt gga acc aaa gatcgg tac tac ata att gat atc aac tac ttc 1202 Glu Leu Gly Thr Lys Asp ArgTyr Tyr Ile Ile Asp Ile Asn Tyr Phe 305 310 315 cca ggt tac ggc aaa atgcca gga tat gag cgc atg ttc aca gac ttc 1250 Pro Gly Tyr Gly Lys Met ProGly Tyr Glu Arg Met Phe Thr Asp Phe 320 325 330 335 tta cta agt ctc gcacaa gca agt aca aaa ggt act taagcgggac 1296 Leu Leu Ser Leu Ala Gln AlaSer Thr Lys Gly Thr 340 345 atgaggtgca aggaagtttg tgaagaccat gctactgacgagatggcata taacggtggc 1356 aggtatgctt ccccaccgcg ccaatgtaca tttgctggagacataagcat aagcgggagg 1416 cttgaggaag ttggcaagtc tcagtgtgtg tgttcaaaatcggtggcaca tgctggactg 1476 gagtaggaaa taaccaagga aacgcttgga tgcgctgtactcatgttgta aaatgtttaa 1536 ctgaatgaac accttcctcg tgatggctcc ctccatcgtaatttggcaac catgagaatt 1596 aattctgcag cttggtaaaa aaaaaaaaaa aaaaaaaaaaaaaaagggcg gccgctcta 1655 4 347 PRT Zea mays 4 Met Arg Leu His Ala GluVal Arg Asp Glu Met Glu Glu Gly Ser Glu 1 5 10 15 Glu Gly Ala Val ThrAla Ser Ala Gly Leu Ser Pro Pro Pro Leu Ile 20 25 30 Gly Ala Ala Ala ProVal Pro Arg Leu Val Val Gly Phe Ala Leu Thr 35 40 45 Lys Lys Lys Val LysSer Phe Leu Gln Pro Lys Leu Leu Leu Leu Ala 50 55 60 Arg Lys Asn Gly IleSer Phe Val Ser Ile Asp Glu Ser Leu Pro Leu 65 70 75 80 Ser Glu Gln GlyPro Phe Asp Val Ile Leu His Lys Ile Thr Arg Lys 85 90 95 Glu Trp Gln LysVal Leu Glu Asp Tyr His Glu Glu His Pro Glu Val 100 105 110 Thr Val LeuAsp Pro Pro Asn Ala Ile Glu His Leu Asn Asn Arg Gln 115 120 125 Ser MetLeu Glu Glu Val Ala Asp Leu Asn Leu Ser Asn Phe Tyr Gly 130 135 140 GluVal Cys Ile Pro Arg Gln Leu Val Ile Thr Lys Asp Pro Ser Ser 145 150 155160 Ile Pro Thr Ser Val Ala Met Ala Gly Leu Thr Leu Pro Leu Val Ala 165170 175 Lys Pro Leu Val Val Asp Gly Thr Ser Lys Gly His Glu Leu Tyr Leu180 185 190 Ala Tyr Asp Glu Ala Ser Leu Ser Met Leu Asp Pro Pro Leu ValLeu 195 200 205 Gln Glu Phe Ile Asn His Gly Gly Ile Leu Phe Lys Val TyrIle Ile 210 215 220 Gly Glu Thr Ile Gln Val Val Arg Arg Phe Ser Leu ProAsp Val Asn 225 230 235 240 Thr Tyr Asp Leu Leu Asn Asn Val Gly Ile TyrArg Leu Pro Arg Val 245 250 255 Ser Cys Ala Ala Ala Ser Ala Asp Asp AlaAsp Leu Asp Pro Leu Ile 260 265 270 Ala Glu Leu Pro Pro Arg Pro Leu LeuGlu Lys Leu Gly Arg Glu Leu 275 280 285 Arg Gly Arg Leu Gly Leu Arg LeuPhe Asn Ile Asp Met Ile Arg Glu 290 295 300 Leu Gly Thr Lys Asp Arg TyrTyr Ile Ile Asp Ile Asn Tyr Phe Pro 305 310 315 320 Gly Tyr Gly Lys MetPro Gly Tyr Glu Arg Met Phe Thr Asp Phe Leu 325 330 335 Leu Ser Leu AlaGln Ala Ser Thr Lys Gly Thr 340 345 5 1600 DNA Zea mays CDS(223)...(1278) 5 gcaccattat taacagctcc gcggtccctc cctccctcgg tcggtcggcgtcggtccctc 60 tccctcccca cccagttagt cctcagccta tcccgtgccc gcgcagagcaccgcctctgc 120 tcgacccacc accctctgtg cagagttaat taaccttgag gtttccgattgcccttccct 180 tccgttcctc tcgcccattc gcggcgagat tcagcggcaa gg atg cgcctg cac 234 Met Arg Leu His 1 gcg gag gtg cgg gat gag atg gag gag gggagc gag gtg ggg gct gtg 282 Ala Glu Val Arg Asp Glu Met Glu Glu Gly SerGlu Val Gly Ala Val 5 10 15 20 acg gct tcg gcg ggg ctg tcg cca ccg ccactc atc ggt gcg gcg gcg 330 Thr Ala Ser Ala Gly Leu Ser Pro Pro Pro LeuIle Gly Ala Ala Ala 25 30 35 ccg gtt ccc cgg ata gtg gtg ggg ttc gcc ctcacg aag aag aag gtg 378 Pro Val Pro Arg Ile Val Val Gly Phe Ala Leu ThrLys Lys Lys Val 40 45 50 aag agc ttc ctg cag ccc aag ctg ctc ctg ctg gccagg aag aat gga 426 Lys Ser Phe Leu Gln Pro Lys Leu Leu Leu Leu Ala ArgLys Asn Gly 55 60 65 atc agt ttt gta tct att gat gag tct ctt ccc ctc tcagaa caa ggc 474 Ile Ser Phe Val Ser Ile Asp Glu Ser Leu Pro Leu Ser GluGln Gly 70 75 80 cct ttt gat gtt att tta cac aag att act agg aag gag tggcag aag 522 Pro Phe Asp Val Ile Leu His Lys Ile Thr Arg Lys Glu Trp GlnLys 85 90 95 100 gtt ctg gag gac tat cac gaa gaa cat cca gaa gtt act gtcctt gac 570 Val Leu Glu Asp Tyr His Glu Glu His Pro Glu Val Thr Val LeuAsp 105 110 115 cca cca aat gct atc gag cat ctg aac aat cga caa tca atgctt gaa 618 Pro Pro Asn Ala Ile Glu His Leu Asn Asn Arg Gln Ser Met LeuGlu 120 125 130 gaa gta gct gat ttg aac ctg tca aat ttc tat gga gaa gtttgt atc 666 Glu Val Ala Asp Leu Asn Leu Ser Asn Phe Tyr Gly Glu Val CysIle 135 140 145 cca cgc cag ctg gtc att acg aaa gat cca tcc tct ata ccaact tct 714 Pro Arg Gln Leu Val Ile Thr Lys Asp Pro Ser Ser Ile Pro ThrSer 150 155 160 gta gct atg gct gga cta act ttg ccc ttg gtt gcc aag ccattg gtt 762 Val Ala Met Ala Gly Leu Thr Leu Pro Leu Val Ala Lys Pro LeuVal 165 170 175 180 gtt gat ggg acg tct aaa ggt cat gaa cta tat ctt gcatat gac gag 810 Val Asp Gly Thr Ser Lys Gly His Glu Leu Tyr Leu Ala TyrAsp Glu 185 190 195 gca tcc ttg tca atg ctt gat ccg cct ctg gtt cta caggaa ttc ata 858 Ala Ser Leu Ser Met Leu Asp Pro Pro Leu Val Leu Gln GluPhe Ile 200 205 210 aac cat ggc ggg atc ctc ttt aag gtg tat atc att ggtgaa aca ata 906 Asn His Gly Gly Ile Leu Phe Lys Val Tyr Ile Ile Gly GluThr Ile 215 220 225 cag gtt gtc cgc agg ttc tct ctt cct gat gtt aac acatat gac tta 954 Gln Val Val Arg Arg Phe Ser Leu Pro Asp Val Asn Thr TyrAsp Leu 230 235 240 cta aac aac gtt ggc atc tat cga ttg cca aga gtt tcatgt gct gca 1002 Leu Asn Asn Val Gly Ile Tyr Arg Leu Pro Arg Val Ser CysAla Ala 245 250 255 260 gct agt gcg gat gat gca gat ctt gac cct ctt attgca gag ctt cct 1050 Ala Ser Ala Asp Asp Ala Asp Leu Asp Pro Leu Ile AlaGlu Leu Pro 265 270 275 cca agg cca ctt cta gag aaa ctg ggc agg gag cttcgt ggc cgg ctt 1098 Pro Arg Pro Leu Leu Glu Lys Leu Gly Arg Glu Leu ArgGly Arg Leu 280 285 290 ggt ttg aga ttg ttc aat ata gat atg att aga gaactt gga acc aaa 1146 Gly Leu Arg Leu Phe Asn Ile Asp Met Ile Arg Glu LeuGly Thr Lys 295 300 305 gat cgg tac tac ata att gat atc aac tac ttc ccaggt tac ggc aaa 1194 Asp Arg Tyr Tyr Ile Ile Asp Ile Asn Tyr Phe Pro GlyTyr Gly Lys 310 315 320 atg cca gga tat gag cgc atg ttc aca gac ttc ttacta agt ctc gca 1242 Met Pro Gly Tyr Glu Arg Met Phe Thr Asp Phe Leu LeuSer Leu Ala 325 330 335 340 caa agc aag tac aaa agg tac tta agc ggg acgtga ggtgcaagga 1288 Gln Ser Lys Tyr Lys Arg Tyr Leu Ser Gly Thr * 345350 agtttgtgaa gaccatgcta ctgacgagat ggcatataac ggtggcagct atgcttcccc1348 accgcgccaa tgtacatttg ctggagacat aagcataagc cggaggcttg aggaagttgg1408 caagtctcag tgtgtgtgtt caaaatcggt ggcacatgct ggactggagt aggaaataac1468 caaggaaacg cttggatgcg ctgtacccat gttgtaaaat gtttaactga atgaacacct1528 tcctcgtgat ggctccctcc atcgtaattt ggcaaccatg agaattaatt ctgcaaaaaa1588 aaaaaaaaaa aa 1600 6 351 PRT Zea mays 6 Met Arg Leu His Ala Glu ValArg Asp Glu Met Glu Glu Gly Ser Glu 1 5 10 15 Val Gly Ala Val Thr AlaSer Ala Gly Leu Ser Pro Pro Pro Leu Ile 20 25 30 Gly Ala Ala Ala Pro ValPro Arg Ile Val Val Gly Phe Ala Leu Thr 35 40 45 Lys Lys Lys Val Lys SerPhe Leu Gln Pro Lys Leu Leu Leu Leu Ala 50 55 60 Arg Lys Asn Gly Ile SerPhe Val Ser Ile Asp Glu Ser Leu Pro Leu 65 70 75 80 Ser Glu Gln Gly ProPhe Asp Val Ile Leu His Lys Ile Thr Arg Lys 85 90 95 Glu Trp Gln Lys ValLeu Glu Asp Tyr His Glu Glu His Pro Glu Val 100 105 110 Thr Val Leu AspPro Pro Asn Ala Ile Glu His Leu Asn Asn Arg Gln 115 120 125 Ser Met LeuGlu Glu Val Ala Asp Leu Asn Leu Ser Asn Phe Tyr Gly 130 135 140 Glu ValCys Ile Pro Arg Gln Leu Val Ile Thr Lys Asp Pro Ser Ser 145 150 155 160Ile Pro Thr Ser Val Ala Met Ala Gly Leu Thr Leu Pro Leu Val Ala 165 170175 Lys Pro Leu Val Val Asp Gly Thr Ser Lys Gly His Glu Leu Tyr Leu 180185 190 Ala Tyr Asp Glu Ala Ser Leu Ser Met Leu Asp Pro Pro Leu Val Leu195 200 205 Gln Glu Phe Ile Asn His Gly Gly Ile Leu Phe Lys Val Tyr IleIle 210 215 220 Gly Glu Thr Ile Gln Val Val Arg Arg Phe Ser Leu Pro AspVal Asn 225 230 235 240 Thr Tyr Asp Leu Leu Asn Asn Val Gly Ile Tyr ArgLeu Pro Arg Val 245 250 255 Ser Cys Ala Ala Ala Ser Ala Asp Asp Ala AspLeu Asp Pro Leu Ile 260 265 270 Ala Glu Leu Pro Pro Arg Pro Leu Leu GluLys Leu Gly Arg Glu Leu 275 280 285 Arg Gly Arg Leu Gly Leu Arg Leu PheAsn Ile Asp Met Ile Arg Glu 290 295 300 Leu Gly Thr Lys Asp Arg Tyr TyrIle Ile Asp Ile Asn Tyr Phe Pro 305 310 315 320 Gly Tyr Gly Lys Met ProGly Tyr Glu Arg Met Phe Thr Asp Phe Leu 325 330 335 Leu Ser Leu Ala GlnSer Lys Tyr Lys Arg Tyr Leu Ser Gly Thr 340 345 350 7 1428 DNA Zea maysCDS (56)...(1084) 7 ccacgcgtcc gcaaatttca atctccatcg atcgattcctcccgaacccg acccg atg 58 Met 1 gcc tcc gac gcc gcc gcc gag ccc tcc tccggc gtc acc cac ccc ccg 106 Ala Ser Asp Ala Ala Ala Glu Pro Ser Ser GlyVal Thr His Pro Pro 5 10 15 cgc tac gtc atc ggt tac gcg ctc gcg ccg aagaag cag caa agc ttc 154 Arg Tyr Val Ile Gly Tyr Ala Leu Ala Pro Lys LysGln Gln Ser Phe 20 25 30 atc cag ccg tcg ctg gtg gcc cag gcg gcg tcg cggggc atg gac ctc 202 Ile Gln Pro Ser Leu Val Ala Gln Ala Ala Ser Arg GlyMet Asp Leu 35 40 45 gtc ccc gtg gat gcg tcg cag ccc ctg gca gag caa gggccc ttc cac 250 Val Pro Val Asp Ala Ser Gln Pro Leu Ala Glu Gln Gly ProPhe His 50 55 60 65 ctc ctc atc cac aag ctc tac gga gac gac tgg cgc gcccag ctc gtg 298 Leu Leu Ile His Lys Leu Tyr Gly Asp Asp Trp Arg Ala GlnLeu Val 70 75 80 gcc ttc gcc gcg cgc cac ccg gcc gtc ccc atc gtc gac ccgccc cac 346 Ala Phe Ala Ala Arg His Pro Ala Val Pro Ile Val Asp Pro ProHis 85 90 95 gcc atc gac cgc ctc cac aac cgc atc tcc atg ctc cag gtc gtctcc 394 Ala Ile Asp Arg Leu His Asn Arg Ile Ser Met Leu Gln Val Val Ser100 105 110 gag ctc gac cac gcc gcc gac cag gac agc act ttc ggt atc cccagc 442 Glu Leu Asp His Ala Ala Asp Gln Asp Ser Thr Phe Gly Ile Pro Ser115 120 125 cag gtc gtc gtc tac gac gct gcc gcg ctc gcc gac ttc gga ctcctt 490 Gln Val Val Val Tyr Asp Ala Ala Ala Leu Ala Asp Phe Gly Leu Leu130 135 140 145 gcc gcg ctc cgc ttc ccg ctc atc gcc aag ccc ctc gtc gccgac ggc 538 Ala Ala Leu Arg Phe Pro Leu Ile Ala Lys Pro Leu Val Ala AspGly 150 155 160 acc gcc aag tcc cac aag atg tcg ctc gtc tac cac cgc gagggc ctc 586 Thr Ala Lys Ser His Lys Met Ser Leu Val Tyr His Arg Glu GlyLeu 165 170 175 ggc aag ctc cgc ccg ccg ctt gtg ctc cag gag ttc gtc aaccat ggc 634 Gly Lys Leu Arg Pro Pro Leu Val Leu Gln Glu Phe Val Asn HisGly 180 185 190 ggc gtc atc ttc aag gtc tac gtc gtc ggc ggc cac gtc acttgc gtc 682 Gly Val Ile Phe Lys Val Tyr Val Val Gly Gly His Val Thr CysVal 195 200 205 aag cgc cgt agc ctg ccc gac gtg tcc ccc gag gat gac gcatcg gcc 730 Lys Arg Arg Ser Leu Pro Asp Val Ser Pro Glu Asp Asp Ala SerAla 210 215 220 225 cag gga tcc gtc tcc ttc tcc cag gtc tcc aac ctc cccact gag cgc 778 Gln Gly Ser Val Ser Phe Ser Gln Val Ser Asn Leu Pro ThrGlu Arg 230 235 240 acg gcg gag gag tac tac ggc gaa aag agt ctc gag gacgcc gtc gtg 826 Thr Ala Glu Glu Tyr Tyr Gly Glu Lys Ser Leu Glu Asp AlaVal Val 245 250 255 ccg ccc gcc gca ttc atc aac cag atc gcg ggc ggc ctccgc cgc gcg 874 Pro Pro Ala Ala Phe Ile Asn Gln Ile Ala Gly Gly Leu ArgArg Ala 260 265 270 ctg ggc ctg caa ctc ttc aac ttc gac atg atc cgc gacgtc cgc gcc 922 Leu Gly Leu Gln Leu Phe Asn Phe Asp Met Ile Arg Asp ValArg Ala 275 280 285 ggc gac cgc tat ctc gtc att gac atc aac tac ttc ccgggc tac gcc 970 Gly Asp Arg Tyr Leu Val Ile Asp Ile Asn Tyr Phe Pro GlyTyr Ala 290 295 300 305 aag atg cca gga tac gag act gtc ctc acg gat ttcttc tgg gag atg 1018 Lys Met Pro Gly Tyr Glu Thr Val Leu Thr Asp Phe PheTrp Glu Met 310 315 320 gtc cat aag gac ggc gtg ggc aac caa cag gag gagaaa ggg gcc aac 1066 Val His Lys Asp Gly Val Gly Asn Gln Gln Glu Glu LysGly Ala Asn 325 330 335 cat gtt gtc gtg aaa taa gatgatgatt gatggcactggatatctggc 1114 His Val Val Val Lys * 340 gaatgctgct gattctggatgcagaattcg atgaggggat ttagttggtt gtagtatctg 1174 gcgaatgctg ctggttctggatgcagaatt tgatgagggg atttagttgg atttcaaccc 1234 atagcatgcc gaggacctcctagctctttc caaaccagtt gtttaggtat cttttctggg 1294 taagtcagct tcatctagtttagtctgtct gaacaaaaga gtgggacatg acccaaacgg 1354 aattctaatg aaaaacgagctctctatctg caaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1414 aaaaaaaaaa aaaa 1428 8342 PRT Zea mays 8 Met Ala Ser Asp Ala Ala Ala Glu Pro Ser Ser Gly ValThr His Pro 1 5 10 15 Pro Arg Tyr Val Ile Gly Tyr Ala Leu Ala Pro LysLys Gln Gln Ser 20 25 30 Phe Ile Gln Pro Ser Leu Val Ala Gln Ala Ala SerArg Gly Met Asp 35 40 45 Leu Val Pro Val Asp Ala Ser Gln Pro Leu Ala GluGln Gly Pro Phe 50 55 60 His Leu Leu Ile His Lys Leu Tyr Gly Asp Asp TrpArg Ala Gln Leu 65 70 75 80 Val Ala Phe Ala Ala Arg His Pro Ala Val ProIle Val Asp Pro Pro 85 90 95 His Ala Ile Asp Arg Leu His Asn Arg Ile SerMet Leu Gln Val Val 100 105 110 Ser Glu Leu Asp His Ala Ala Asp Gln AspSer Thr Phe Gly Ile Pro 115 120 125 Ser Gln Val Val Val Tyr Asp Ala AlaAla Leu Ala Asp Phe Gly Leu 130 135 140 Leu Ala Ala Leu Arg Phe Pro LeuIle Ala Lys Pro Leu Val Ala Asp 145 150 155 160 Gly Thr Ala Lys Ser HisLys Met Ser Leu Val Tyr His Arg Glu Gly 165 170 175 Leu Gly Lys Leu ArgPro Pro Leu Val Leu Gln Glu Phe Val Asn His 180 185 190 Gly Gly Val IlePhe Lys Val Tyr Val Val Gly Gly His Val Thr Cys 195 200 205 Val Lys ArgArg Ser Leu Pro Asp Val Ser Pro Glu Asp Asp Ala Ser 210 215 220 Ala GlnGly Ser Val Ser Phe Ser Gln Val Ser Asn Leu Pro Thr Glu 225 230 235 240Arg Thr Ala Glu Glu Tyr Tyr Gly Glu Lys Ser Leu Glu Asp Ala Val 245 250255 Val Pro Pro Ala Ala Phe Ile Asn Gln Ile Ala Gly Gly Leu Arg Arg 260265 270 Ala Leu Gly Leu Gln Leu Phe Asn Phe Asp Met Ile Arg Asp Val Arg275 280 285 Ala Gly Asp Arg Tyr Leu Val Ile Asp Ile Asn Tyr Phe Pro GlyTyr 290 295 300 Ala Lys Met Pro Gly Tyr Glu Thr Val Leu Thr Asp Phe PheTrp Glu 305 310 315 320 Met Val His Lys Asp Gly Val Gly Asn Gln Gln GluGlu Lys Gly Ala 325 330 335 Asn His Val Val Val Lys 340 9 1295 DNA Zeamays CDS (19)...(1143) 9 cgatagtcca ccaagtca atg gcg gcg gag cag tgc cagtcc tca ggc ggc 51 Met Ala Ala Glu Gln Cys Gln Ser Ser Gly Gly 1 5 10agc tcg ccg cgg cct cgc gcc gca tac acc atc ggc tac gcg atg ctg 99 SerSer Pro Arg Pro Arg Ala Ala Tyr Thr Ile Gly Tyr Ala Met Leu 15 20 25 cccaac aag cac gat acc ttc gtc cag ccg tcg ttc atc gac ctg gca 147 Pro AsnLys His Asp Thr Phe Val Gln Pro Ser Phe Ile Asp Leu Ala 30 35 40 gcg cagcac ggc atc cgg ctc gtg gcg ctc gac gcc tcc agg ccg ctc 195 Ala Gln HisGly Ile Arg Leu Val Ala Leu Asp Ala Ser Arg Pro Leu 45 50 55 gcg gag cagggc ccc cag ctg gac ctc gtc gtg cac aag ctg tac ggc 243 Ala Glu Gln GlyPro Gln Leu Asp Leu Val Val His Lys Leu Tyr Gly 60 65 70 75 cag gcg tggcgc gcg cgg ctg gag gcc ttc tcg gcg ctc cac ccg gac 291 Gln Ala Trp ArgAla Arg Leu Glu Ala Phe Ser Ala Leu His Pro Asp 80 85 90 gtc cca atc atcgac ccg ccc gcc gcc atc gac cgc atc ctg gac cgc 339 Val Pro Ile Ile AspPro Pro Ala Ala Ile Asp Arg Ile Leu Asp Arg 95 100 105 ttc acc atg ctggac gtc gtc tcg ggg ctc gac tgc gtg gcc gtg ccc 387 Phe Thr Met Leu AspVal Val Ser Gly Leu Asp Cys Val Ala Val Pro 110 115 120 agg cag gtc atggtc cac gac gcc ggg gcc ctg cag cag gcc gcc gac 435 Arg Gln Val Met ValHis Asp Ala Gly Ala Leu Gln Gln Ala Ala Asp 125 130 135 gcc gcc gcc gacgac gtg ctc ggc ctc ggc ggc ctc cgg ttc ccg ctc 483 Ala Ala Ala Asp AspVal Leu Gly Leu Gly Gly Leu Arg Phe Pro Leu 140 145 150 155 gtc gcc aagccc gtg gag gtg gac ggc agc gcg gcg tcg cac gac ctc 531 Val Ala Lys ProVal Glu Val Asp Gly Ser Ala Ala Ser His Asp Leu 160 165 170 tgc ctg gtgtac cgc cgc gag ggc ctg cgc ggc ctg cgc ggc cgc ccg 579 Cys Leu Val TyrArg Arg Glu Gly Leu Arg Gly Leu Arg Gly Arg Pro 175 180 185 ccg ctc gtgctg cag gag ttc gcc aac cac ggc ggc gtg ctc ttc aag 627 Pro Leu Val LeuGln Glu Phe Ala Asn His Gly Gly Val Leu Phe Lys 190 195 200 gtg tac gtggtg ggc gac cgc gcc acg tgc gtg gtg cgg agc agc ctg 675 Val Tyr Val ValGly Asp Arg Ala Thr Cys Val Val Arg Ser Ser Leu 205 210 215 ccg gac gtgccg ccg gag cgc ctc cgg gac ccc gcc gcc gcc gcc gcg 723 Pro Asp Val ProPro Glu Arg Leu Arg Asp Pro Ala Ala Ala Ala Ala 220 225 230 235 gcc cccttc gcc aac atc tcc ctc ctc gcc ccc agc ggc ggc gac gag 771 Ala Pro PheAla Asn Ile Ser Leu Leu Ala Pro Ser Gly Gly Asp Glu 240 245 250 ggc tccgag aag gtg gta ccg ccg ccc cag gac ttc gtc gac agg gtc 819 Gly Ser GluLys Val Val Pro Pro Pro Gln Asp Phe Val Asp Arg Val 255 260 265 gcc cgcgag atc cgg cgg gca gtg ggc ctg cac ctc atc aac ttc gac 867 Ala Arg GluIle Arg Arg Ala Val Gly Leu His Leu Ile Asn Phe Asp 270 275 280 ctc atccgg acg agg gac gac gct gca ggc ggc gac gcc aat aag tac 915 Leu Ile ArgThr Arg Asp Asp Ala Ala Gly Gly Asp Ala Asn Lys Tyr 285 290 295 ctc gtcctc gac atc aac tac tgc ccg ggc tac tcc aaa atg ccc ggc 963 Leu Val LeuAsp Ile Asn Tyr Cys Pro Gly Tyr Ser Lys Met Pro Gly 300 305 310 315 tttgag cct gtc ctc act gaa ttc ttc ctg gag agg ctt cgc tct cgc 1011 Phe GluPro Val Leu Thr Glu Phe Phe Leu Glu Arg Leu Arg Ser Arg 320 325 330 agcaga agc atc gat gag cgg cct gcc ccg ggg gcg gag gcg agg cag 1059 Ser ArgSer Ile Asp Glu Arg Pro Ala Pro Gly Ala Glu Ala Arg Gln 335 340 345 gcagag gca gag gca gag gcc gag ccc agc agc gcc acc atc ccc atc 1107 Ala GluAla Glu Ala Glu Ala Glu Pro Ser Ser Ala Thr Ile Pro Ile 350 355 360 ccgccg gga gcg gag gcg agg ctg gct cag gcc taa attcgccagg 1153 Pro Pro GlyAla Glu Ala Arg Leu Ala Gln Ala * 365 370 ttcctcacat catccagtttgtttaatttg gaccatatac accagtgaag cgtgaagtga 1213 agccgccttg attctaacctttcattgcaa agggaattaa ataaacacca gttgctttgt 1273 acccaaaaaa aaaaaaaaaaaa 1295 10 374 PRT Zea mays 10 Met Ala Ala Glu Gln Cys Gln Ser Ser GlyGly Ser Ser Pro Arg Pro 1 5 10 15 Arg Ala Ala Tyr Thr Ile Gly Tyr AlaMet Leu Pro Asn Lys His Asp 20 25 30 Thr Phe Val Gln Pro Ser Phe Ile AspLeu Ala Ala Gln His Gly Ile 35 40 45 Arg Leu Val Ala Leu Asp Ala Ser ArgPro Leu Ala Glu Gln Gly Pro 50 55 60 Gln Leu Asp Leu Val Val His Lys LeuTyr Gly Gln Ala Trp Arg Ala 65 70 75 80 Arg Leu Glu Ala Phe Ser Ala LeuHis Pro Asp Val Pro Ile Ile Asp 85 90 95 Pro Pro Ala Ala Ile Asp Arg IleLeu Asp Arg Phe Thr Met Leu Asp 100 105 110 Val Val Ser Gly Leu Asp CysVal Ala Val Pro Arg Gln Val Met Val 115 120 125 His Asp Ala Gly Ala LeuGln Gln Ala Ala Asp Ala Ala Ala Asp Asp 130 135 140 Val Leu Gly Leu GlyGly Leu Arg Phe Pro Leu Val Ala Lys Pro Val 145 150 155 160 Glu Val AspGly Ser Ala Ala Ser His Asp Leu Cys Leu Val Tyr Arg 165 170 175 Arg GluGly Leu Arg Gly Leu Arg Gly Arg Pro Pro Leu Val Leu Gln 180 185 190 GluPhe Ala Asn His Gly Gly Val Leu Phe Lys Val Tyr Val Val Gly 195 200 205Asp Arg Ala Thr Cys Val Val Arg Ser Ser Leu Pro Asp Val Pro Pro 210 215220 Glu Arg Leu Arg Asp Pro Ala Ala Ala Ala Ala Ala Pro Phe Ala Asn 225230 235 240 Ile Ser Leu Leu Ala Pro Ser Gly Gly Asp Glu Gly Ser Glu LysVal 245 250 255 Val Pro Pro Pro Gln Asp Phe Val Asp Arg Val Ala Arg GluIle Arg 260 265 270 Arg Ala Val Gly Leu His Leu Ile Asn Phe Asp Leu IleArg Thr Arg 275 280 285 Asp Asp Ala Ala Gly Gly Asp Ala Asn Lys Tyr LeuVal Leu Asp Ile 290 295 300 Asn Tyr Cys Pro Gly Tyr Ser Lys Met Pro GlyPhe Glu Pro Val Leu 305 310 315 320 Thr Glu Phe Phe Leu Glu Arg Leu ArgSer Arg Ser Arg Ser Ile Asp 325 330 335 Glu Arg Pro Ala Pro Gly Ala GluAla Arg Gln Ala Glu Ala Glu Ala 340 345 350 Glu Ala Glu Pro Ser Ser AlaThr Ile Pro Ile Pro Pro Gly Ala Glu 355 360 365 Ala Arg Leu Ala Gln Ala370 11 1837 DNA Zea mays CDS (97)...(1617) misc_feature (1)...(1837) n =A,T,C or G 11 gggcgttcgg cccggcagcc cccaagtccc atcgccgagc agaaagtcaggaacagaact 60 caggcgttgg cgattggcat ctccctcccc taagcc atg gct acc gggcgg ccc 114 Met Ala Thr Gly Arg Pro 1 5 gta cga ctc gtg ctg gat gcc tccctc ctc ctc gac ccc tcc tcc acc 162 Val Arg Leu Val Leu Asp Ala Ser LeuLeu Leu Asp Pro Ser Ser Thr 10 15 20 agg gag gcg gcg gcg gtg gcg ctg cggncc ggg gta gag gag ttg ctg 210 Arg Glu Ala Ala Ala Val Ala Leu Arg XaaGly Val Glu Glu Leu Leu 25 30 35 cgg cgg ttg cgc tac tcc aac ctg agc gtggca atc tgc tat gca gag 258 Arg Arg Leu Arg Tyr Ser Asn Leu Ser Val AlaIle Cys Tyr Ala Glu 40 45 50 ggc atg cca act aat gag tca gac ttt ctt gaaaag gtc gca agc tca 306 Gly Met Pro Thr Asn Glu Ser Asp Phe Leu Glu LysVal Ala Ser Ser 55 60 65 70 cac ttg ttt gga tct ata gta ctt ctt gca aaaagt ggg aat ctt tct 354 His Leu Phe Gly Ser Ile Val Leu Leu Ala Lys SerGly Asn Leu Ser 75 80 85 cca att gaa tta atg ata gaa tgg agc cga aca agtttt tgt ttt tat 402 Pro Ile Glu Leu Met Ile Glu Trp Ser Arg Thr Ser PheCys Phe Tyr 90 95 100 gcg act tca aga gtt gac aaa ggt tta att tct gagctc cag aat cag 450 Ala Thr Ser Arg Val Asp Lys Gly Leu Ile Ser Glu LeuGln Asn Gln 105 110 115 aac tgg aga gtt ctt tct gta gct aat gaa tgt agcata gag gtt cct 498 Asn Trp Arg Val Leu Ser Val Ala Asn Glu Cys Ser IleGlu Val Pro 120 125 130 ggt gtt tta aat gtt caa agg ctt cag gag ttg cttctc acc ttg gct 546 Gly Val Leu Asn Val Gln Arg Leu Gln Glu Leu Leu LeuThr Leu Ala 135 140 145 150 act cta atg aaa aag gaa ctt tgt ggc tca tctgtt ctg gtg att gga 594 Thr Leu Met Lys Lys Glu Leu Cys Gly Ser Ser ValLeu Val Ile Gly 155 160 165 tat ata atg aaa aaa tcc cgt gag gaa gac ttcgca aag gca act tct 642 Tyr Ile Met Lys Lys Ser Arg Glu Glu Asp Phe AlaLys Ala Thr Ser 170 175 180 tta gaa gga gca ttt ccc ata tat cct agt aagggc agt ctt atc ttt 690 Leu Glu Gly Ala Phe Pro Ile Tyr Pro Ser Lys GlySer Leu Ile Phe 185 190 195 gtt ccc ctc tct ttc gaa att cca tta agt ttgcaa ctg caa gaa gtt 738 Val Pro Leu Ser Phe Glu Ile Pro Leu Ser Leu GlnLeu Gln Glu Val 200 205 210 gat atg gtc ctc cac aaa ata act gat gag attgtc aag atc gat cca 786 Asp Met Val Leu His Lys Ile Thr Asp Glu Ile ValLys Ile Asp Pro 215 220 225 230 aac tgc tcc att gat ttt cca aaa ggg atttca ttt tct gca gga atg 834 Asn Cys Ser Ile Asp Phe Pro Lys Gly Ile SerPhe Ser Ala Gly Met 235 240 245 tct gaa att ata aga ttt gtg gaa gag caccct gat ttt tgt atc atg 882 Ser Glu Ile Ile Arg Phe Val Glu Glu His ProAsp Phe Cys Ile Met 250 255 260 gac cca ttt aaa aac att tac cca ttg cttgac cgt ctt caa atc caa 930 Asp Pro Phe Lys Asn Ile Tyr Pro Leu Leu AspArg Leu Gln Ile Gln 265 270 275 aaa atc ctt gtc cgg ttg caa gaa ctt ggcact gaa gga aag cca aaa 978 Lys Ile Leu Val Arg Leu Gln Glu Leu Gly ThrGlu Gly Lys Pro Lys 280 285 290 ctt cga gca ccg tat tct tgc aag gtt gacagt ttt cat aat ggt gaa 1026 Leu Arg Ala Pro Tyr Ser Cys Lys Val Asp SerPhe His Asn Gly Glu 295 300 305 310 ttg gat aag cat cta gta gaa gct aattta tcc ttc cca ctc att gtg 1074 Leu Asp Lys His Leu Val Glu Ala Asn LeuSer Phe Pro Leu Ile Val 315 320 325 aag cca caa gtc gct tgt gga gtc gctgat gcc cac aat atg gca ctg 1122 Lys Pro Gln Val Ala Cys Gly Val Ala AspAla His Asn Met Ala Leu 330 335 340 gtt ttt cag att gaa gaa ttt agc aacctc agt gtg ccc ctt cct gct 1170 Val Phe Gln Ile Glu Glu Phe Ser Asn LeuSer Val Pro Leu Pro Ala 345 350 355 gtg cta cag gaa tac gtg gat cac ggatcc aag att ttc aag ttc tat 1218 Val Leu Gln Glu Tyr Val Asp His Gly SerLys Ile Phe Lys Phe Tyr 360 365 370 gtg atc gga gac aag gtt ttc tac gccgtt aga gac tca atg ccc aac 1266 Val Ile Gly Asp Lys Val Phe Tyr Ala ValArg Asp Ser Met Pro Asn 375 380 385 390 gcg cgc ttc ctg aag ccg tcg tcagga ggt gaa gct ctt aca ttt aat 1314 Ala Arg Phe Leu Lys Pro Ser Ser GlyGly Glu Ala Leu Thr Phe Asn 395 400 405 agt ttg aag act ctt ccg gtg gctacg aat gag cag cga ccg cag acc 1362 Ser Leu Lys Thr Leu Pro Val Ala ThrAsn Glu Gln Arg Pro Gln Thr 410 415 420 ggc gcg gaa gat ggc aag ctg ttagat gcc gat ctg gta gaa gag gcc 1410 Gly Ala Glu Asp Gly Lys Leu Leu AspAla Asp Leu Val Glu Glu Ala 425 430 435 gca aaa ttc ctg aag ggg ctg cttggg ctt aca gta ttt gga ttc gat 1458 Ala Lys Phe Leu Lys Gly Leu Leu GlyLeu Thr Val Phe Gly Phe Asp 440 445 450 gtc gtc gtc caa gaa ggc acc ggagac cat gtc ata gtg gac ctg aac 1506 Val Val Val Gln Glu Gly Thr Gly AspHis Val Ile Val Asp Leu Asn 455 460 465 470 tac ctg ccg tcg ttc aaa gaggtt ccc gac tcg gag gcg gtg cct gcg 1554 Tyr Leu Pro Ser Phe Lys Glu ValPro Asp Ser Glu Ala Val Pro Ala 475 480 485 ttc tgg gac gcg gtc agg caggcg tac gag tcg acg cgc ggg aat gcg 1602 Phe Trp Asp Ala Val Arg Gln AlaTyr Glu Ser Thr Arg Gly Asn Ala 490 495 500 aat gcc cag ggt taataaggtgtca aggctcttcc cgaataagtg aatctacgtg 1657 Asn Ala Gln Gly * 505gagtggagcg cagcagagag aggagagccg cagtggttgt aatggttctg gagcagactc 1717ggagtaatgt tcggctgtag ctgtgggaat aagcgaaatc gggagcggaa taataattaa 1777caacaatccg ccatgtttag ctgtccaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 183712 506 PRT Zea mays UNSURE (1)...(506) Xaa= any amino acid 12 Met AlaThr Gly Arg Pro Val Arg Leu Val Leu Asp Ala Ser Leu Leu 1 5 10 15 LeuAsp Pro Ser Ser Thr Arg Glu Ala Ala Ala Val Ala Leu Arg Xaa 20 25 30 GlyVal Glu Glu Leu Leu Arg Arg Leu Arg Tyr Ser Asn Leu Ser Val 35 40 45 AlaIle Cys Tyr Ala Glu Gly Met Pro Thr Asn Glu Ser Asp Phe Leu 50 55 60 GluLys Val Ala Ser Ser His Leu Phe Gly Ser Ile Val Leu Leu Ala 65 70 75 80Lys Ser Gly Asn Leu Ser Pro Ile Glu Leu Met Ile Glu Trp Ser Arg 85 90 95Thr Ser Phe Cys Phe Tyr Ala Thr Ser Arg Val Asp Lys Gly Leu Ile 100 105110 Ser Glu Leu Gln Asn Gln Asn Trp Arg Val Leu Ser Val Ala Asn Glu 115120 125 Cys Ser Ile Glu Val Pro Gly Val Leu Asn Val Gln Arg Leu Gln Glu130 135 140 Leu Leu Leu Thr Leu Ala Thr Leu Met Lys Lys Glu Leu Cys GlySer 145 150 155 160 Ser Val Leu Val Ile Gly Tyr Ile Met Lys Lys Ser ArgGlu Glu Asp 165 170 175 Phe Ala Lys Ala Thr Ser Leu Glu Gly Ala Phe ProIle Tyr Pro Ser 180 185 190 Lys Gly Ser Leu Ile Phe Val Pro Leu Ser PheGlu Ile Pro Leu Ser 195 200 205 Leu Gln Leu Gln Glu Val Asp Met Val LeuHis Lys Ile Thr Asp Glu 210 215 220 Ile Val Lys Ile Asp Pro Asn Cys SerIle Asp Phe Pro Lys Gly Ile 225 230 235 240 Ser Phe Ser Ala Gly Met SerGlu Ile Ile Arg Phe Val Glu Glu His 245 250 255 Pro Asp Phe Cys Ile MetAsp Pro Phe Lys Asn Ile Tyr Pro Leu Leu 260 265 270 Asp Arg Leu Gln IleGln Lys Ile Leu Val Arg Leu Gln Glu Leu Gly 275 280 285 Thr Glu Gly LysPro Lys Leu Arg Ala Pro Tyr Ser Cys Lys Val Asp 290 295 300 Ser Phe HisAsn Gly Glu Leu Asp Lys His Leu Val Glu Ala Asn Leu 305 310 315 320 SerPhe Pro Leu Ile Val Lys Pro Gln Val Ala Cys Gly Val Ala Asp 325 330 335Ala His Asn Met Ala Leu Val Phe Gln Ile Glu Glu Phe Ser Asn Leu 340 345350 Ser Val Pro Leu Pro Ala Val Leu Gln Glu Tyr Val Asp His Gly Ser 355360 365 Lys Ile Phe Lys Phe Tyr Val Ile Gly Asp Lys Val Phe Tyr Ala Val370 375 380 Arg Asp Ser Met Pro Asn Ala Arg Phe Leu Lys Pro Ser Ser GlyGly 385 390 395 400 Glu Ala Leu Thr Phe Asn Ser Leu Lys Thr Leu Pro ValAla Thr Asn 405 410 415 Glu Gln Arg Pro Gln Thr Gly Ala Glu Asp Gly LysLeu Leu Asp Ala 420 425 430 Asp Leu Val Glu Glu Ala Ala Lys Phe Leu LysGly Leu Leu Gly Leu 435 440 445 Thr Val Phe Gly Phe Asp Val Val Val GlnGlu Gly Thr Gly Asp His 450 455 460 Val Ile Val Asp Leu Asn Tyr Leu ProSer Phe Lys Glu Val Pro Asp 465 470 475 480 Ser Glu Ala Val Pro Ala PheTrp Asp Ala Val Arg Gln Ala Tyr Glu 485 490 495 Ser Thr Arg Gly Asn AlaAsn Ala Gln Gly 500 505 13 1808 DNA Zea mays CDS (72)...(1580) 13gaattcggca cgagcagaaa gccaggaaca gaactcaggc gttggcgatt ggcatctccc 60tcccctaagc c atg gct acc ggg cgg ccc gta cga ctc gtg ctg gat gcc 110 MetAla Thr Gly Arg Pro Val Arg Leu Val Leu Asp Ala 1 5 10 tcc ctc ctc ctcgac ccc tcc tcc acc agg gag gcg gcg gcg gtg gcg 158 Ser Leu Leu Leu AspPro Ser Ser Thr Arg Glu Ala Ala Ala Val Ala 15 20 25 ctg cgg ccc ggg gtagag gag ctg ctg cgg cgg ttg cgc tac tcc aac 206 Leu Arg Pro Gly Val GluGlu Leu Leu Arg Arg Leu Arg Tyr Ser Asn 30 35 40 45 ctg aac gtg gca atctgc tat gca gag ggc atg cca aat aat gag tca 254 Leu Asn Val Ala Ile CysTyr Ala Glu Gly Met Pro Asn Asn Glu Ser 50 55 60 ggc ttt ctt gaa aag gtcgca agc tca cac ttg ttt ggc tct att gca 302 Gly Phe Leu Glu Lys Val AlaSer Ser His Leu Phe Gly Ser Ile Ala 65 70 75 ctt ctt gcg aaa agc ggg aatctt tct cta act gaa tta atg tta gaa 350 Leu Leu Ala Lys Ser Gly Asn LeuSer Leu Thr Glu Leu Met Leu Glu 80 85 90 tgg agc cga aca agt ttt tgt ttttat gcg acg tca aga gtt gac aaa 398 Trp Ser Arg Thr Ser Phe Cys Phe TyrAla Thr Ser Arg Val Asp Lys 95 100 105 ggt tta att tct gag ctc cag aatcag aac tgg aga gtt ctt tct gta 446 Gly Leu Ile Ser Glu Leu Gln Asn GlnAsn Trp Arg Val Leu Ser Val 110 115 120 125 gct aat gaa tgt agc ata gaggtt cct ggt gtt tta aat gtt caa agg 494 Ala Asn Glu Cys Ser Ile Glu ValPro Gly Val Leu Asn Val Gln Arg 130 135 140 ctt cag cag ttg ctt ctc accttg gct act cta ata aaa agg gaa cta 542 Leu Gln Gln Leu Leu Leu Thr LeuAla Thr Leu Ile Lys Arg Glu Leu 145 150 155 tgt gac tca tct gtt ctg gtgatt gga tat ata atg aaa aaa tcc cgt 590 Cys Asp Ser Ser Val Leu Val IleGly Tyr Ile Met Lys Lys Ser Arg 160 165 170 gag gaa gac ttc gca agg agagga gca ttt ccc ata tat cct agt aag 638 Glu Glu Asp Phe Ala Arg Arg GlyAla Phe Pro Ile Tyr Pro Ser Lys 175 180 185 ggc agt ctt atc ttt gtt cccctc tct ttt gaa ctt cct tta agt ttg 686 Gly Ser Leu Ile Phe Val Pro LeuSer Phe Glu Leu Pro Leu Ser Leu 190 195 200 205 caa ctg caa gaa gtt gatatg gcc ctc cac aaa ata acc gat gag att 734 Gln Leu Gln Glu Val Asp MetAla Leu His Lys Ile Thr Asp Glu Ile 210 215 220 gtc aag att gat cca aactgc tcc att gat ttt cca aaa ggg att tca 782 Val Lys Ile Asp Pro Asn CysSer Ile Asp Phe Pro Lys Gly Ile Ser 225 230 235 ttt tct aca gga atg tctgaa att ata agg ttt gtg gaa gag cac cct 830 Phe Ser Thr Gly Met Ser GluIle Ile Arg Phe Val Glu Glu His Pro 240 245 250 gat ttc cgc atc atg gatcca ttt aaa aac att tac cca ttg ctt gat 878 Asp Phe Arg Ile Met Asp ProPhe Lys Asn Ile Tyr Pro Leu Leu Asp 255 260 265 cgt ctt caa atc caa aaaatc ctt gtc cgg ttg caa gaa ctt ggc att 926 Arg Leu Gln Ile Gln Lys IleLeu Val Arg Leu Gln Glu Leu Gly Ile 270 275 280 285 gaa gga aag cca aaactt cga gca ccg tat tct tgc aag gtt gac aat 974 Glu Gly Lys Pro Lys LeuArg Ala Pro Tyr Ser Cys Lys Val Asp Asn 290 295 300 ttt gat aat ggt gaattg gat aag cat cta gca gaa gct aat tta tcc 1022 Phe Asp Asn Gly Glu LeuAsp Lys His Leu Ala Glu Ala Asn Leu Ser 305 310 315 ttc cca ctc att gtgaag cca caa gtt gct tgt gga gtc gct gat gcc 1070 Phe Pro Leu Ile Val LysPro Gln Val Ala Cys Gly Val Ala Asp Ala 320 325 330 cac aat atg gca ctggtt ttt cag att gaa gaa ttt agc aac ctc agt 1118 His Asn Met Ala Leu ValPhe Gln Ile Glu Glu Phe Ser Asn Leu Ser 335 340 345 gtg ccc ctt cct gctgtg cta cag gaa tac gtg gat cac gga tcc aag 1166 Val Pro Leu Pro Ala ValLeu Gln Glu Tyr Val Asp His Gly Ser Lys 350 355 360 365 att ttc aag ttctat gtg atc gga gac aag gtt ttc tac gcc gtt aga 1214 Ile Phe Lys Phe TyrVal Ile Gly Asp Lys Val Phe Tyr Ala Val Arg 370 375 380 gac tca atg cccaac gcg cgc ttc ctt aag ccg tcg tca gga ggt gaa 1262 Asp Ser Met Pro AsnAla Arg Phe Leu Lys Pro Ser Ser Gly Gly Glu 385 390 395 gct ctt aca tttaat agt ttg aag act ctt ccg gtg gct acc aat gag 1310 Ala Leu Thr Phe AsnSer Leu Lys Thr Leu Pro Val Ala Thr Asn Glu 400 405 410 cag cga ccg cagacc gcc gcg gaa gat ggc aag ctg tta gat gcc gat 1358 Gln Arg Pro Gln ThrAla Ala Glu Asp Gly Lys Leu Leu Asp Ala Asp 415 420 425 ctg gta gaa gaggcc gca aaa ttc ctg aag ggg ctg ctt ggg ctt aca 1406 Leu Val Glu Glu AlaAla Lys Phe Leu Lys Gly Leu Leu Gly Leu Thr 430 435 440 445 gta ttt ggattc gat gtc gtc gtc caa gaa ggc acc gga gac cat gtc 1454 Val Phe Gly PheAsp Val Val Val Gln Glu Gly Thr Gly Asp His Val 450 455 460 ata gtg gacctg aac tac ctg ccg tcg ttc aaa gag gtt ccc aac tcg 1502 Ile Val Asp LeuAsn Tyr Leu Pro Ser Phe Lys Glu Val Pro Asn Ser 465 470 475 gag gcg gtgcct gca ttc tgg gac gcg gtc agg cag gcg tgc gag tcg 1550 Glu Ala Val ProAla Phe Trp Asp Ala Val Arg Gln Ala Cys Glu Ser 480 485 490 acg cgc gggaat gcg aat gtc cag ggt taa cctcaatgat cttcccgaat 1600 Thr Arg Gly AsnAla Asn Val Gln Gly * 495 500 aataagtgaa tctacctgga gcgtagcagagaggagagcc gcagtggtgt tcactggttg 1660 taatggtcag ctgtagctgt gggaataagtgaaatacaat ccgccaagtt tagctgtcga 1720 tctcgtcgcc gtggtgtatt ctgtcacgatgtcagtttca tgtgaatctg ctaactgatg 1780 gtttcccaaa aaaaaaaaaa aaaaaaaa1808 14 502 PRT Zea mays 14 Met Ala Thr Gly Arg Pro Val Arg Leu Val LeuAsp Ala Ser Leu Leu 1 5 10 15 Leu Asp Pro Ser Ser Thr Arg Glu Ala AlaAla Val Ala Leu Arg Pro 20 25 30 Gly Val Glu Glu Leu Leu Arg Arg Leu ArgTyr Ser Asn Leu Asn Val 35 40 45 Ala Ile Cys Tyr Ala Glu Gly Met Pro AsnAsn Glu Ser Gly Phe Leu 50 55 60 Glu Lys Val Ala Ser Ser His Leu Phe GlySer Ile Ala Leu Leu Ala 65 70 75 80 Lys Ser Gly Asn Leu Ser Leu Thr GluLeu Met Leu Glu Trp Ser Arg 85 90 95 Thr Ser Phe Cys Phe Tyr Ala Thr SerArg Val Asp Lys Gly Leu Ile 100 105 110 Ser Glu Leu Gln Asn Gln Asn TrpArg Val Leu Ser Val Ala Asn Glu 115 120 125 Cys Ser Ile Glu Val Pro GlyVal Leu Asn Val Gln Arg Leu Gln Gln 130 135 140 Leu Leu Leu Thr Leu AlaThr Leu Ile Lys Arg Glu Leu Cys Asp Ser 145 150 155 160 Ser Val Leu ValIle Gly Tyr Ile Met Lys Lys Ser Arg Glu Glu Asp 165 170 175 Phe Ala ArgArg Gly Ala Phe Pro Ile Tyr Pro Ser Lys Gly Ser Leu 180 185 190 Ile PheVal Pro Leu Ser Phe Glu Leu Pro Leu Ser Leu Gln Leu Gln 195 200 205 GluVal Asp Met Ala Leu His Lys Ile Thr Asp Glu Ile Val Lys Ile 210 215 220Asp Pro Asn Cys Ser Ile Asp Phe Pro Lys Gly Ile Ser Phe Ser Thr 225 230235 240 Gly Met Ser Glu Ile Ile Arg Phe Val Glu Glu His Pro Asp Phe Arg245 250 255 Ile Met Asp Pro Phe Lys Asn Ile Tyr Pro Leu Leu Asp Arg LeuGln 260 265 270 Ile Gln Lys Ile Leu Val Arg Leu Gln Glu Leu Gly Ile GluGly Lys 275 280 285 Pro Lys Leu Arg Ala Pro Tyr Ser Cys Lys Val Asp AsnPhe Asp Asn 290 295 300 Gly Glu Leu Asp Lys His Leu Ala Glu Ala Asn LeuSer Phe Pro Leu 305 310 315 320 Ile Val Lys Pro Gln Val Ala Cys Gly ValAla Asp Ala His Asn Met 325 330 335 Ala Leu Val Phe Gln Ile Glu Glu PheSer Asn Leu Ser Val Pro Leu 340 345 350 Pro Ala Val Leu Gln Glu Tyr ValAsp His Gly Ser Lys Ile Phe Lys 355 360 365 Phe Tyr Val Ile Gly Asp LysVal Phe Tyr Ala Val Arg Asp Ser Met 370 375 380 Pro Asn Ala Arg Phe LeuLys Pro Ser Ser Gly Gly Glu Ala Leu Thr 385 390 395 400 Phe Asn Ser LeuLys Thr Leu Pro Val Ala Thr Asn Glu Gln Arg Pro 405 410 415 Gln Thr AlaAla Glu Asp Gly Lys Leu Leu Asp Ala Asp Leu Val Glu 420 425 430 Glu AlaAla Lys Phe Leu Lys Gly Leu Leu Gly Leu Thr Val Phe Gly 435 440 445 PheAsp Val Val Val Gln Glu Gly Thr Gly Asp His Val Ile Val Asp 450 455 460Leu Asn Tyr Leu Pro Ser Phe Lys Glu Val Pro Asn Ser Glu Ala Val 465 470475 480 Pro Ala Phe Trp Asp Ala Val Arg Gln Ala Cys Glu Ser Thr Arg Gly485 490 495 Asn Ala Asn Val Gln Gly 500 15 26 DNA Artificial Sequenceforward primer for itpk 15 attcctcccg aacccgaccc gatggc 26 16 25 DNAArtificial Sequence reverse primer for itpk 16 cggaattcta atgaaaaacgagctc 25 17 22 DNA Artificial Sequence reverse primer for itpk 17caaccatgtt gtcgtgaaat aa 22 18 48 DNA Artificial Sequence PCR primer tocreate SmaI site for itpk-2 18 tatcacccgg gatggtgtcg ggcgtgtgcgtggggacgga ggggcagg 48 19 38 DNA Artificial Sequence PCR primer tocreate NotI site for itpk2 19 atctagtaac ggtgcggccg cccgagtagt ctcctcgc38 20 41 DNA Artificial Sequence PCR primer to create SmaI site foritpk3 20 actcgtaaca tgaagccacc cgggatgcgc ggtgcacgcg g 41 21 40 DNAArtificial Sequence PCR primer to create NotI site for itpk3 21ctagtaacgg tgcggccgct taagtacctt ttgtacttgc 40 22 26 DNA ArtificialSequence reverse primer for itpk5 22 catcttattt cacgacaaca tggttg 26 2327 DNA Artificial Sequence TUSC primer for itpk5 23 ccgaagaagcagcaaagctt catccag 27 24 27 DNA Artificial Sequence TUSC primer foritpk5 24 tggtttggaa agagctagga ggtcctc 27 25 32 DNA Artificial SequenceTUSC Mu primer 25 agagaagcca acgccawcgc ctcyatttcg tc 32 26 36 DNAArtificial Sequence Sal A-20 poly A primer 26 tcgacccacg cgtccgaaaaaaaaaaaaaa aaaaaa 36 27 24 DNA Artificial Sequence reverse primer forITPK 27 agctcgtttt tcattagaat tccg 24 28 1364 DNA Zea mays CDS(34)...(138) 28 gaattcgccc ttattcctcc cgaacccgac ccg atg gcc tcc gac gccgcc gcc 54 Met Ala Ser Asp Ala Ala Ala 1 5 gag ccc tcc tcc ggc gtc acccac ccc ccg cgc tac gtc atc ggt tac 102 Glu Pro Ser Ser Gly Val Thr HisPro Pro Arg Tyr Val Ile Gly Tyr 10 15 20 gcg ctc gcg ccg aag aag cag cagagc ttc atc tag ccgtcgctgg 148 Ala Leu Ala Pro Lys Lys Gln Gln Ser PheIle * 25 30 tggcccaggc ggcgtcgcgg ggcatggacc tcgtccccgt ggatgcgtcgcagcccctcg 208 cagagcaagg gcccttccac ctcctcatcc acaagctcta cggagacgactggcgcgccc 268 agctcgtggc cttcgccgcg cgccacccgg ccgtccccat cgtcgacccgccccacgcca 328 tcgaccgcct ccacaaccgc atctccatgc tccaggtcgt ctccgagctcgaccacgccg 388 ccgaccagga cagcactttc ggtatcccca gccaggtcgt cgtctacgacgccgccgcgc 448 tcgccgactt cggactcctt gccgcgctcc gcttcccgct catcgccaagcccctcgtcg 508 ccgacggcac cgccaagtcc cacaagatgt cgctcgtcta ccaccgcgagggcctcggca 568 agctccgccc gccgcttgtg ctccaggagt tcgtcaacca tggcggcgtcatcttcaagg 628 tctacgtcgt cggcggccac gtcacttgcg tcaagcgccg tagccttcccgacgtgtccc 688 ccgaggatga cgcatcggcc cagggatccg tctccttctc ccaggtctccaacctcccca 748 ctgagcgcac ggcggaggag tactacggcg aaaagagtct cgaggacgccgtcgtgccgc 808 ccgccgcatt catcaaccag atcgcgggcg gcctccgccg cgcgctgggcctgcaactct 868 tcaacttcga catgatccgc gacgtccgcg ccggcgaccg ctatctcgtcattgacatca 928 actacttccc gggctacgcc aagatgccag gatacgagac tgtcctcacggatttcttct 988 gggagatggt ccatgaggac ggcgtgggca accaacagga ggagaaaggggccaaccatg 1048 ttgtcgtgaa ataagatgat gattgatggc actggatatc tggcgaatgctgctgattct 1108 ggatgcagaa ttcgatgagg ggatttagtt ggttgtagta tctggcgaatgctgctggtt 1168 ctggatgcag aatttgatga ggggatttag ttggatttca acccacagcatgccgaggac 1228 ctcctagctc tttccagacc agttgtttag gtatcttttc tgggtaagtcagcttcatct 1288 agtttagtct gtctgaacaa aagagtggga catgacccra acggaattctatgaaaaacg 1348 agctaagggc gaattc 1364 29 34 PRT Zea mays 29 Met Ala SerAsp Ala Ala Ala Glu Pro Ser Ser Gly Val Thr His Pro 1 5 10 15 Pro ArgTyr Val Ile Gly Tyr Ala Leu Ala Pro Lys Lys Gln Gln Ser 20 25 30 Phe Ile

What is claimed is:
 1. An isolated nucleic acid comprising a memberselected from the group consisting of: (a) a polynucleotide having atleast 87% sequence identity to SEQ ID NO: 1, or 93% sequence identity toSEQ ID NO: 7, or 80% identity to SEQ ID NOS: 9, 11, or 13; wherein the %sequence identity is determined over the entire length of the codingsequence by the GAP algorithm using default parameters; (b) apolynucleotide which encodes a polypeptide of SEQ ID NOS: 2, 8, 10, 12,or 14; (c) a polynucleotide which selectively hybridizes, understringent conditions and a wash in 0.1×SSC at 60° C., to apolynucleotide of SEQ ID NOS: 1, 7, 9, 11, or 13; (d) a polynucleotideamplified from a plant nucleic acid library using the primers of SEQ IDNOS: 15, 16, 17, 22, and 27 or primers determined by using Vector NTISuite, InforMax Version 5, or an equivalent method; (e) a polynucleotidehaving the sequence set forth in SEQ ID NOS:1, 7, 9, 11, or 13; (f) apolynucleotide comprising at least 45 contiguous nucleotides of SEQ IDNOS: 9, 11, or 13; and (g) a polynucleotide complementary to apolynucleotide of (a) through (f) wherein the isolated nucleic acidmodulates the level of ITPK.
 2. The isolated nucleic acid of claim 1,wherein the polynucleotide is from a monocot or dicot.
 3. A vectorcomprising at least one nucleic acid of claim
 1. 4. An expressioncassette comprising at least one nucleic acid of claim 1 operably linkedto a promoter, wherein the nucleic acid is in sense or antisenseorientation.
 5. The expression cassette of claim 4, wherein the nucleicacid is operably linked in antisense orientation to the promoter.
 6. Anon-human host cell containing at least one nucleic acid of claim
 1. 7.The host cell of claim 6, wherein the host cell is a plant cell.
 8. Atransformed plant comprising at least one nucleic acid of claim
 1. 9.The transformed plant of claim 8, wherein the plant is corn, barley,soybean, sorghum, wheat, rice, alfalfa, safflower, sunflower, canola,cotton, or millet.
 10. A transformed seed from the transformed plant ofclaim
 8. 11. A method for modulating inositol 1,3,4-trisphosphate5/6-kinase (ITPK) activity or levels in a host cell, comprising: (a)transforming a host cell with at least one expression cassette of claim4; and (b) growing the transformed host cell to modulate ITPK activityin the host cell.
 12. The method of claim 11, wherein the host cell is aplant cell.
 13. The method of claim 12, wherein the plant cell is from amonocot or a dicot.
 14. The method of claim 12, further comprisingproducing a transformed plant from the plant cell.
 15. A transformedplant produced by the method of claim
 14. 16. The transformed plant ofclaim 15, wherein the plant is corn, barley, soybean, sorghum, wheat,rice, alfalfa, safflower, sunflower, canola, cotton, or millet.
 17. Atransformed seed from the plant of claim
 15. 18. The method of claim 12wherein the level of phytate is reduced.
 19. The method of claim 12wherein the level of non-phytate phosphorous is increased.
 20. A methodof improving the nutritional value of animal feed, comprising: (a)transforming a plant host cell with at least one expression cassette ofclaim 4 to reduce phytate content; (b) growing the transformed host cellto modulate ITPK activity in the host cell; (c) generating a plant withthe transformed genotype; and (d) producing animal feed from the plant,wherein the animal feed has improved nutritional value.
 21. The methodof claim 20, wherein the plant cell is from a monocot or a dicot.
 22. Atransformed plant produced by the method of claim
 20. 23. A transformedseed from the plant of claim
 22. 24. The transformed plant of claim 22,wherein the plant is corn, barley, soybean, sorghum, wheat, rice,safflower, sunflower, canola, or millet.
 25. The method of claim 20,wherein the level of non-phytate phosphorous is increased.
 26. A methodof decreasing the level of phosphorous in non-ruminant animal wastecomprising providing said non-ruminant animal said animal feed producedby the method of claim
 20. 27. A method of increasing the level ofavailable phosphorous in animal feed, comprising: (a) transforming aplant host cell with at least one expression cassette of claim 4 toreduce phytate content; (b) growing the transformed host cell tomodulate ITPK activity in the host cell; (c) generating a plant with thetransformed genotype; and (d) producing animal feed from the plant,wherein the animal feed has an increased level of available phosphorous.28. The method of claim 27, wherein the plant cell is from a monocot ora dicot.
 29. A transformed plant produced by the method of claim
 27. 30.A transformed seed from the plant of claim
 29. 31. The transformed plantof claim 29, wherein the plant is corn, barley, soybean, sorghum, wheat,rice, safflower, sunflower, or canola.
 32. A method of decreasing thelevel of phosphorous in non-ruminant animal waste comprising providingsaid non-ruminant animal said animal feed produced by the method ofclaim
 27. 33. A method of altering plant phenotype comprising: (a)transforming a plant host cell with at least one ITPK polynucleotide ofclaim 1 and at least one polynucleotide of interest; (b) growing thetransformed host cell to modulate the activity of ITPK and thepolynucleotide of interest in the host cell; and (c) generating atransformed plant with an altered phenotype.
 34. The method of claim 33,wherein the activity of ITPK is downregulated and wherein the activityof the polynucleotide of interest is up-regulated.
 35. The method ofclaim 34, wherein the polynucleotide of interest is myo-inositolmonophosphatase (IMP) or phytase.
 36. The method of claim 33, whereinthe activity of ITPK and the activity of the polynucleotide of interestare each downregulated.
 37. The method of claim 36, wherein thepolynucleotide of interest is inositol polyphosphate kinase (IPPK) ormyo-inositol 1-phosphate synthase (MI 1PS).
 38. A transformed plantproduced by the method of claim
 33. 39. The transformed plant of claim38, wherein the plant is corn, barley, soybean, sorghum, wheat, rice,alfalfa, safflower, sunflower, canola, cotton, or millet.
 40. Atransformed seed from the plant of claim
 38. 41. An isolated proteincomprising a member selected from the group consisting of: (a) apolypeptide comprising at least 25 contiguous amino acids of SEQ ID NOS:10, 12, or 14; (b) a polypeptide comprising at least 94% sequenceidentity compared to the full-length of SEQ ID NOS: 2 or 8, or 75%sequence identity compared to the full-length of SEQ ID NOS: 10, 12, or14; wherein the percent sequence identity is based on the entiresequence length and is determined by the GAP algorithm using defaultparameters; (c) a polypeptide encoded by the nucleic acid of claim 1;(d) a polypeptide encoded by a nucleic acid of SEQ ID NOS:1, 7, 9, 11 or13; and (e) a polypeptide having the sequence set forth in SEQ ID NOS:2, 8, 10, 12, or
 14. 42. An isolated ribonucleic acid sequence encodingthe protein of claim 41.